JP2011109936A - Chemically modified ribonucleotide synthesis and gene expression inhibiting effect for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide: an siRNA having a high gene-inhibiting effect, which is double-stranded RNA having target gene expression inhibiting ability and 21 to 27 bases and is used in RNA interference methods; and a method for efficiently inhibiting gene expression using the siRNA. <P>SOLUTION: There are disclosed: an siRNA preparing method in which a low molecular weight compound having an unsaturated hydrocarbon compound as a basic skeleton is conveniently, efficiently and chemically modified to ribonucleotide by known methods such as a phosphoroamidite method or an active esterification method using a succinimide linker; and a method for efficiently inhibiting gene expression that is represented by an RNA interference method using the siRNA. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

    The present invention relates to a method for synthesizing chemically modified ribonucleotides and the effect of suppressing gene expression using them.

      The genome information of various organisms is being clarified one after another by the results of recent genome projects, etc., and research to elucidate gene functions such as replication, transcription, and translation of individual genes based on this genome information is remarkable. It is developing. In such efforts to elucidate functions, DNA or RNA is introduced into cells or animal individuals, and the target gene is overexpressed to activate the function, or conversely, the function is stopped by deletion or deletion. A technique for collecting information on various phenotypic changes and elucidating the functions of genes and proteins, for example, by reducing them, is widely used.

      Numerous gene function analysis methods are currently being developed, and various methods are selected according to the purpose. Since then, 1. No complicated operation like gene knockout method is required 2. Gene function can be examined by using base sequence information and low molecular weight RNA 3. Number compared with antisense method etc. The technology is used by many researchers aiming at gene function analysis because it has a suppressive activity 10 to several hundred times. As one of the above small RNAs, a chemically synthesized double-stranded ribonucleotide (hereinafter referred to as siRNA) having an oligo length of about 21 to 27 bases is used. There are cases in which higher functionality is desired for reasons such as improvement in sex, and as one of the means, chemical modification to ribonucleotides is performed.

    For example, in order to improve the inhibitory effect, the introduction of the cell membrane permeable peptide or the like increases the introduction effect, or if the effect is sustained, the 2nd hydroxyl group of the sugar chain ribose is increased for the purpose of increasing the resistance to RNase. In some cases, a method such as substitution is effective, and the selectivity to an organ is improved by applying a lipid compound such as cholesterol.

However, with regard to peptide-modified ribonucleotides, there are cases where the yield of the peptide itself, the efficiency of modification to ribonucleotides, etc. are inadequate. There is a case. In addition, although the 2-position hydroxyl group-substituted ribonucleotide can be expected to have an excellent effect in terms of enzyme resistance, it has been reported that the RNAi effect depends on the position and number of mononucleotides to be substituted. Some cases are not suitable as siRNA.
A. Fire, S. Xu, MK. Montgomery, SA. Kostas, SE. Driver, CC. Mello, Nature, 391, 806 (1998). F. Simeoni, MC Morris, F. Heitz, G. Divita, Nucleic Acids Res., 31, 2717 (2003) YLChiu, TM Rana, RNA, 9, 1034 (2003) J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, RK Pandey, T. Racie, KG Rajeev, I. Rohl, I. Toudjarska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan, HP Vornlocher, Nature 432, 173 (2004)

      An object of the present invention is to provide a siRNA having a high gene suppression effect using a chemically modified ribonucleotide obtained by a simple and efficient method for chemically modifying ribonucleotides.

      As a result of intensive research on the above problems, the present inventor has found a covalent conjugate of a low molecular unsaturated hydrocarbon compound such as pyrene or 3,7-dimethyl-octa-2,6-diene and ribonucleotide. Giving a simple and efficient method using phosphoramidite method on phase or active esterification method via succinimide linker, etc., and the resulting chemically modified siRNA consisting of chemically modified ribonucleotide and its complementary strand has high gene suppression effect As a result, the present invention has been completed.

    That is, according to the present invention, a composition in which a low molecular weight unsaturated hydrocarbon compound represented by the following general formula (1) is bound to at least one arbitrary nucleotide in a ribonucleotide sequence, directly or via a linker. siRNA is provided.

In the formula, X is a hydrogen atom, a halogen group, a sulfonyl group, Y is a hydrogen atom, a halogen group, a nitro group, a sulfonyl group, Z is a hydrogen atom, a halogen group, a hydroxyl group, or a sulfonyl group. In particular, derivatives in which X, Y and Z are hydrogen atoms are preferred.

The siRNA is also provided as a composition in which a low molecular weight unsaturated hydrocarbon compound represented by the following general formula (2) is bound to at least one of ribonucleotides directly or via a linker.

    In the formula, A is a hydrogen atom, a saturated alkyl chain, or an unsaturated alkyl chain, B is a hydrogen atom, a saturated alkyl chain, or an unsaturated alkyl chain, and the alkyl group has a chain length of 0 to 20, preferably 5 -15, more preferably 5-10. In one embodiment of the present invention, derivatives wherein X is a methyl group and Y is 2-methyl-pent-2-ene are preferred.

In one embodiment of the present invention, when the ribonucleotide is provided as a composition obtained by binding a low molecular weight unsaturated hydrocarbon compound and ribonucleotide via a linker, the linker includes the following general formulas (3) to (3) to ( 12).


In the formula, X is any one of O, S, SS, CO, CS, NH, CH ₂ , COO, CONH, NHCO, NHCONH, OCO ₂ , OCONH, NHCSO, NHCSNH, OCSNH, -O-PO ₂ -O-. However, n in the formula (3) represents an integer of 1 to 20, preferably 1 to 10, and more preferably 1 to 5. N in the formula (4) represents an integer of 0 to 20, preferably 0 to 10, more preferably 0 to 5.

      In addition, the siRNA comprising the chemically modified ribonucleotide provided by the chemical modification method of the present invention can be used in a reduced amount due to its suppression effect, and a gene analysis method excellent in accuracy and efficiency is provided. obtain.

    According to the present invention, a chemically modified ribonucleotide having a high gene expression suppression effect in RNAi and the like is obtained through a phosphoramidite method of an unsaturated hydrocarbon-based low molecular weight compound derivative and a ribonucleotide, or a succinimide linker. It was possible to synthesize the siRNA excellent in performance at low cost by simply and efficiently synthesizing on the solid phase by an active esterification method or the like. In addition, the suppression effect of the siRNA can be reduced by reducing the amount used, thereby reducing the load on the cells and improving the analysis accuracy, or reducing the cost per analysis and analyzing more samples. To provide an efficient genetic analysis method. This is because of the serious impact on the current health care system caused by changes in the social structure due to the trend of long life and declining birthrate in Japan. It can be one of useful materials and methods in efforts to reduce health risks through prevention and early treatment.

It is a figure which shows the HPLC analysis result of chemically modified ribonucleotide (Example 1). It is a figure which shows the PAGE analysis result of siRNA which consists of an unmodified and chemically modified ribonucleotide (Example 1). It is the graph which compared the mRNA expression level derived from the Lamin gene in NIH3T3 cell by the real-time PCR method in the RNA interference method using chemically modified ribonucleotide (Example 1). It is the graph which compared the expression level of the protein (Lamin A / C) derived from the Lamin gene in the HeLa cell by the western blotting method in the RNA interference method using chemically modified ribonucleotide (Example 1).

    The chemically modified ribonucleotide provided by the present invention exhibits a suppression effect superior to that of unmodified in suppression of gene expression based on RNA interference method, and is a pyrene derivative or 3,7-dimethyl-octa-2, A 21-27 base double-stranded ribostructure characterized in that at least one of the unsaturated hydrocarbon compounds having a 6-diene derivative or the like is a composition bonded to an arbitrary position of a ribonucleotide directly or via a linker It is a nucleotide.

    The present inventor has found a ribonucleotide that can be useful as an analysis of genetic information or as a nucleic acid drug by causing the gene expression suppression effect by using the chemically modified ribonucleotide. That is, in the presence of the novel ribonucleotide, gene expression is effectively suppressed by the gene expression suppression method for RNAi and the like. Therefore, the amount used can be reduced as compared with conventional unmodified siRNA, and it can be an effective gene analysis method in terms of analysis accuracy and analysis cost.

Examples of the derivative modified with ribonucleotide according to the present invention include compounds represented by the following general formula (1) or (2).

    In the formula, X is a hydrogen atom, halogen group, sulfonyl group, Y is a hydrogen atom, halogen group, nitro group, sulfonyl group, Z is a hydrogen atom, halogen group, hydroxyl group, sulfonyl group, A is a hydrogen atom, saturated alkyl An unsaturated hydrocarbon compound derivative having a hydrogen atom, a saturated alkyl chain, an unsaturated alkyl chain, or the like is used for B.

    According to the present invention, it is possible to effectively suppress the expression of a target gene in the RNA interference method in the presence of the siRNA targeting the mRNA derived from the target gene. Expression suppression and its analysis method can be performed by combining with techniques already known in the art, such as the method described in the paper such as Elbashir, SM, et.al, Nature, 411: 494-498, 2001. Analysis results can be provided.

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to the following examples.

1. Synthesis of pyrene-modified ribonucleotide (Example 1)
A method for synthesizing the siRNA when pyrene is used as the unsaturated hydrocarbon compound represented by the general formula (1) to be modified to the ribonucleotide of the present invention will be described.

The specific synthesis is first described in MH Caruthers, et. Al., Tetrahedron Lett., 22, 1859 (1981) or SL Beaucage, et. Al., Current Protocols in Nucleic Acid Chemistry., Vol. 1, pp. 3.3.1-3.3.20, John Wiley & Sons (2000), a desired ribonucleotide base sequence was synthesized by an automatic nucleic acid synthesizer (H-8 manufactured by Geneworld) by a known phosphoramidite method. As a sequence, a sequence targeting the Lamin gene (nuclear membrane protein gene) was selected, and a sense strand and an antisense strand required for RNAi were respectively synthesized. Next, when preparing a ribonucleotide modified with a pyrene derivative at the 5 terminus of the sense strand, the 5-terminal DMT (Dimethoxytrityl) group of the unmodified ribonucleotide obtained in the above synthesis is deprotected and prepared in advance in a synthesizer. 1- (10b, 10c-Dihydro-pyren-1-ylmethyl) -pyrrolidin phosphoramidite (Glen Research 10-1987-90) was used and modified by the same synthesis method. This ribonucleotide can also be modified by an active esterification method using a succinimide linker, by an amide bond between a carbon chain 10 carboxylic acid succinimidyl linker (Glen Research) and aminopyrene. Can be obtained.

Lamin-siRNA sense (* Pyrene modified)
(5⇒3) * AAC UGG ACUU CCA GAA GAA CAT T
Lamin-siRNA antisense
(5⇒3) UGU UCU UCU GGA AGU CCA GTT

    The purity of the obtained unpurified pyrene-modified ribonucleotide was measured by HPLC. Lichrosorb RP-18 (4.6 × 250 mm, GL science) was used as the analytical column. Use eluent A / 0.1M TEAA (10% MeCN, pH 7.0) and eluent B / 0.1M TEAA (50% MeCN, pH 7.0) as the eluent. A linear gradient was set such that the ratio of liquid B was 0% to 100% over 40 min. After the analysis, HPLC purification was performed under the same HPLC conditions, and a part of the obtained sample was analyzed. FIG. 1A is an analysis result of unpurified pyrene-modified ribonucleotide. FIG. 1B is an analysis result of pyrene-modified ribonucleotide after purification.

2. Synthesis of pyrene-modified siRNA Each pyrene-modified ribonucleotide (sense strand) and its complementary strand (antisense strand) prepared in 5 x annealing buffer (2.5 g K-acetate, 1.8 g HEPES, 0.11 g Mg-acetate 4 water) The solution was mixed and dissolved in Japanese, 2.0 mL 2 M KOH, RNase Free Water 50 mL to make a 20 μM concentration solution. After treatment at 90 ° C. for 1 minute, the mixture was cooled to 37 ° C. over 1 hour and treated at 4 ° C. for 30 minutes to obtain the target siRNA. FIG. 2A shows the results of polyacrylamide gel electrophoresis analysis of siRNA composed of unmodified ribonucleotides and FIG. 2B.

3. Verification of gene suppression effect using pyrene-modified siRNA Trypsin-treated NIH3T3 cells or HeLa cells are dispersed in DMEM (Invitrogen) medium (10% fetal bovine serum (FBS), 1% penicillin-streptomycin), 12-well plate The cells were seeded with 7 × 10 ⁴ cells per well and cultured at 37 ° C. for 24 hours (medium amount was 2 mL / well). Next, opti-MEM (manufactured by Invitrogen) 100 μl so that each final concentration after adding a medium diluted siRNA (20 μM) 10 to 200 times becomes 0.1, 0.2, 0.5, 2, 5, 10, 20 nM Add to the mixture solution of Lipofectamin RNAiMAX (Invitrogen) 1.6 μl and opti-MEM 100 μl, which is the introduction agent, and incubate at room temperature for 15 to 20 minutes to the medium of each well at 37 ° C. Incubated for 72 hours. In addition, a negative control siRNA and an unmodified Lamin-siRNA (see the sequence below) to be compared were also prepared by the same synthesis method described above and added to the medium of each well by the same method.

Lamin-siRNA sense (non-modified)
(5⇒3) CUG GAC UUC CAG AAG AAC ATT
Lamin-siRNA antisense
(5⇒3) UGU UCU UCU GGA AGU CCA GTT

Control-siRNA sense
(5⇒3) UUC UCC GAA CGU GUC ACG UTT
Control-siRNA antisense
(5⇒3) ACG UGA CAC GUU CGG AGA ATT

    After incubation, RNA was extracted from NIH3T3 cells using RNeasy Mini Kit (Qiagen), and target mRNA-derived cDNA was prepared using Takara RNA PCR Kit (AMV) Ver 3.0 (Takara). Subsequently, the mRNA expression suppression rate of the target Lamin gene was confirmed by Real-time PCR Mx 3005P (Stratagene). As a result, the siRNA provided by the present invention has a suppressive effect as high as about 65% in the control-siRNA ratio and about 15% in the unmodified Lamin-siRNA ratio even in a concentration range that is about 100 times lower than the normal protocol. Indicated. FIG. 3 is a graph comparing the mRNA expression levels derived from the Lamin gene in NIH3T3 cells by the real-time PCR method in the RNA interference method using the siRNA.

    After incubation, proteins are extracted from HeLa cells using RIPA buffer (PIERCE), Western-blotting method using Lamin A / C antibody (BD biosciences) as the primary antibody and HRP-labeled antibody (CALBIOCHEM) as the secondary antibody. Was used to confirm the Lamin A / C protein expression inhibitory effect. As a result, the siRNA provided by the present invention showed a better expression-suppressing effect than the data of Control-siRNA or unmodified Lamin-siRNA even in a concentration range about 100 times lower than that of the normal protocol. FIG. 4 is a graph comparing the expression level of Lamin gene-derived protein (Lamin A / C) in HeLa cells by the Western blotting method in the RNA interference method using the siRNA.

    A series of results showed that by using a known amidite method or the like, an unsaturated hydrocarbon compound can be easily and efficiently chemically modified into ribonucleotides. Moreover, the siRNA provided by the present invention showed an expression suppression effect equivalent to or higher than that of the unmodified one in the gene expression suppression effect using the RNA interference method, and its usefulness was proved.

The siRNA provided by the present invention can be provided in a chemical modification method for improving gene expression suppression without using a complicated method or an expensive reagent as found in conventional synthesis methods. Can be used as an effective gene expression suppression tool. Therefore, it is expected to be used in the medical industry such as various test drugs, diagnostic drugs, and nucleic acid drugs.

Claims (5)

    In a gene expression suppression method such as RNA interference method, it is a double-stranded RNA (siRNA) having 21 to 27 bases that suppresses the expression of a target gene, and is directly or directly on at least one of the nucleotides in the sequence The siRNA comprising a chemically modified ribonucleotide obtained by modifying an unsaturated hydrocarbon compound via a linker. The siRNA according to claim 1, comprising a ribonucleotide obtained by modifying a pyrene derivative represented by the following general formula (1) as an unsaturated hydrocarbon compound.



A pyrene derivative in which X is a hydrogen atom, halogen group, sulfonyl group, Y is a hydrogen atom, halogen group, nitro group, sulfonyl group, and Z is a hydrogen atom, halogen group, hydroxyl group, sulfonyl group. The siRNA according to claim 1, comprising a ribonucleotide obtained by modifying an unsaturated alkyl derivative represented by the following general formula (2) as an unsaturated hydrocarbon compound.



An unsaturated alkyl derivative in which A is a hydrogen atom, a saturated alkyl chain, or an unsaturated alkyl chain, and B is a hydrogen atom, a saturated alkyl chain, an unsaturated alkyl chain, or the like.     A method for synthesizing the chemically modified ribonucleotide using the phosphoramidite method or the active esterification method using a succinimide linker, which is the chemically modified ribonucleotide constituting the siRNA according to claims 1 to 3. The gene expression suppression method represented by the RNA interference method etc. which use this siRNA in any one of Claims 1-3.