JP5139067B2 - Efficient novel loop sequences for shRNA expression - Google Patents

Description

  The present invention relates to a method and a series of techniques related to it, which are useful in the fields of medicine, cell engineering, genetic engineering, developmental engineering, etc., and make it possible to improve the effect of RNA gene suppression.

In recent years, gene expression suppression by small RNAs has been discovered one after another. A 21-mer to 23-mer double-stranded RNA called siRNA (short interfering RNA) suppresses gene expression in a sequence-specific manner. Since 1998, Fire et al. (Non-patent Document 1) discovered that double-stranded RNA causes gene silencing in a nematode sequence-specifically, RNA processed by 21-23mer cleaves mRNA. After the mechanism (Non-Patent Document 2) and the presence of RISC (RNA-induced silencing complex) (Non-Patent Document 3) and the cloning of Dicer (Non-Patent Document 4), in 2001, Elbashir et al. It has been proved that sequence-specific expression suppression by siRNA is also possible in cells, and research using siRNA has become very popular.
On the other hand, miRNA (microRNA) that plays a major role in expression control in many stages such as development, moves to the cytoplasm with a short hairpin structure (pre-miRNA), and is cleaved by Dicer to become mature miRNA. It is said that miRNA generally suppresses expression at the translational stage by binding complementarily to the 3 ′ untranslated region.

  In mammalian cells, when siRNA or miRNA is artificially expressed in cells, it is common to express shRNA (short hairpin RNA) from an expression vector. At this time, the loop sequence between the sense sequence and the antisense sequence is said to be important for translocation to the cytoplasm and recognition by Dicer. In recent reports, miRNA-derived sequences are often used as shRNA loop sequences. It is used. However, despite the reports suggesting the importance of loop sequences in shRNA, effective loop sequence searches are rarely performed except for the study by Miyagishi et al. (Non-patent Document 6).

Fire A, 5 others, Nature, 1998, Vol. 391, p. 806-811 Zamore PO, 3 others, Cell, 2000, Vol, 101, p. 25-33 Hammond SM, 3 others, Nature, 2000, Vol. 404, p. 293-296 Bernstein E, 3 others, Nature, 2001, Vol. 409, p. 363-366 Elbashir SM, 5 others, Nature, 2001, Vol. 411, p. 494-498 Miyagi M, 4 others, J. et al. Gene Med. 2004, Vol. 6, p. 715-723

  An object of the present invention is to provide a loop sequence having a high effect in gene suppression using shRNA. If a stronger inhibitory effect can be obtained using the loop sequence of the present invention, it is considered to have a great influence on, for example, gene function analysis experiments. Moreover, in the treatment using gene suppression using shRNA, there is a possibility that the therapeutic effect is influenced.

  As a result of diligent research, the present inventors conducted the following comprehensive loop sequence search and found an effective loop sequence. These novel loop sequences were confirmed to show a higher gene suppression effect than the conventionally used sequences, and the invention was completed. That is, short RNA expressed in human cells was purified, screened using a comprehensive gene expression analysis technique (MPSS) using microbeads, and the sequence was analyzed. In addition, the database is analyzed based on the sequence information, and sequences that do not hit any known miRNA, rRNA, or tRNA are extracted from sequences that match 100% with the human genome, and new microRNA candidates are selected from secondary structure prediction. Obtained. Nine types of sequences were randomly selected from the obtained novel microRNA candidates, and a loop sequence was obtained from the stem loop sequence. An shRNA expression vector containing the loop sequence thus selected was constructed, and the present invention was completed.

That is, the first invention of the present invention relates to a method for screening a shRNA loop sequence useful for gene suppression of a target gene, which comprises the following steps.
(1) a step of extracting low molecular weight RNA from a biological sample,
(2) determining the base sequence of the low molecular weight RNA obtained in (1),
(3) Based on the genome information of the living body, the genome sequences before and after the base sequence obtained in (2) are obtained, and a series of sequences consisting of the base sequence obtained in (2) and the genomic sequences before and after that A step of selecting a sequence corresponding to the following (A) and (B):
(A) a sequence of a region having the lowest free energy per certain length, which forms a stem-loop structure;
(B) a sequence in which the base sequence obtained in (2) is present in the stem region of the stem-loop structure,
(4) A loop sequence is determined from the genome sequence obtained in (3), and a shRNA in which the target gene sequence and the complementary sequence in the opposite direction are connected to both ends of the loop sequence so as to form a stem region is constructed. The process of
(5) A step of confirming the gene suppression effect of the target gene using the shRNA obtained in (4).

  The second invention of the present invention relates to a nucleic acid containing a shRNA loop sequence useful for gene suppression obtained by the method of the first invention of the present invention.

In the second invention of the present invention, the loop sequence may be a sequence selected from a base sequence selected from the following:
(1) the base sequence described in any one of SEQ ID NOS: 12 to 20 in the sequence listing; or (2) one or more bases in the base sequence described in any one of SEQ ID NOS: 12 to 20 in the sequence listing A nucleotide sequence having at least one of deletion, addition, insertion or substitution.

  The third invention of the present invention relates to a vector for expressing shRNA containing the nucleic acid of the second invention of the present invention.

  The fourth invention of the present invention relates to a kit comprising the nucleic acid of the second invention of the present invention or the vector of the third invention of the present invention.

  The shRNA expression vector containing the sequence of the present invention can provide a high gene suppression effect compared with a loop sequence that is often used conventionally.

It is a figure which shows the effect of the screening method and nucleic acid gene suppression of this invention. It is a figure which shows the effect of the screening method and nucleic acid gene suppression of this invention. It is a figure which shows the effect of the screening method and nucleic acid gene suppression of this invention. It is a figure which shows the effect of the screening method and nucleic acid gene suppression of this invention. It is a figure which shows the effect of the screening method and nucleic acid gene suppression of this invention.

  In this specification, “stem loop (hairpin loop) structure” means a double-stranded part (stem) generated by hydrogen bonding between inverted repeats existing on single-stranded RNA or DNA and a loop sandwiched between them. The structure which consists of these parts is shown. That is, in the stem region, the base sequences of the two regions are complementary to each other and exist in opposite directions. The hydrogenated inverted repeats may be completely complementary or partially complementary. The structure of the loop portion may have a stem region in part, and a bulge may be inserted in the stem region. The stem loop structure can be predicted and confirmed by a secondary structure prediction algorithm for nucleic acids. This algorithm is described in, for example, Vienna RNA Package (Hofacker I et al., Nucleic Acids Research, Vol. 31 (13), p. 3429-31 (2003)), MFOLD (Zuker M et al., Nucleic Acids Research, Vol. ), P. 3406-15 (2003)).

  As used herein, “shRNA” refers to RNA having a stem-loop structure. The shRNA is a single-stranded RNA, and inverted repeats present on the single-stranded RNA are annealed to form a double-stranded RNA (double RNA: dsRNA) portion called a stem region. The portion sandwiched between the inverted repeat sequences is called a loop region. The loop region array is called a loop array.

  In this specification, “useful for gene suppression” means that it can be used when gene expression is suppressed, and particularly strongly induces gene suppression, that is, strongly suppresses gene expression. Say. The shRNA loop sequence useful for gene suppression is a shRNA loop sequence that can be used when gene suppression is performed. Although not particularly limited, for example, a sequence generally used for the loop structure of an shRNA expression vector Compared with the loop region of the sequence shown in No. 21 (Brummelkamp et al. Science. 2002 296: 550-553.), A loop sequence having a high effect of gene suppression is particularly useful for gene suppression.

  As used herein, “free energy” is a value given by the Zuker method (Zuker M et al., RNA biochemistry and biotechnology, (Kluwer Academic Publishers), p. 11-43 (1999)), and is a higher order structure of RNA. Used to predict.

1. Screening method of shRNA loop sequence useful for gene suppression The screening method of shRNA loop sequence useful for gene suppression of the present invention includes the following steps.
(1) a step of extracting low molecular weight RNA from a biological sample,
(2) determining the base sequence of the low molecular weight RNA obtained in (1),
(3) Based on the genome information of the living body, the genome sequences before and after the base sequence obtained in (2) are obtained, and a series of sequences consisting of the base sequence obtained in (2) and the genomic sequences before and after that A step of selecting a sequence corresponding to the following (A) and (B):
(A) a sequence of a region having the lowest free energy per certain length, which forms a stem-loop structure;
(B) a sequence in which the base sequence obtained in (2) is present in the stem region of the stem-loop structure,
(4) A loop sequence is determined from the genome sequence obtained in (3), and a shRNA in which the target gene sequence and the complementary sequence in the opposite direction are connected to both ends of the loop sequence so as to form a stem region is constructed. The process of
(5) A step of confirming the gene suppression effect of the target gene using the shRNA obtained in (4).

  In the screening method of the present invention, the biological sample is not particularly limited as long as it is a sample considered to contain RNA, and may be derived from any of cultured cells, tissues, organs, body fluids and the like. Furthermore, it is preferably a biological sample derived from a living body that performs gene suppression, but may be a biological sample derived from a living organism other than the living body that performs gene suppression if it is useful for gene suppression. As a method for extracting RNA from these biological samples, a method known to those skilled in the art may be used, and many kits optimized for biological samples are commercially available.

  In the screening method of the present invention, low molecular RNA means RNA having a lower molecular weight than general mRNA, tRNA, and rRNA, and RNA having a length of about miRNA is particularly suitable. Although not particularly limited, for example, RNA having a length of 10 to 100 bases, preferably 15 to 50 bases, more preferably 17 to 30 bases, and particularly preferably 21 to 27 bases. The method for extracting low molecular weight RNA is not particularly limited as long as RNA can be fractionated by molecular weight. Examples thereof include excision by gel electrophoresis, fractionation by column fractionation, and filtration by a filter. .

  As a method for determining the base sequence of the low-molecular RNA, a general RNA sequencing method can be used. Although not particularly limited, for example, cDNA is synthesized from RNA, and chain termination method, MPSS method (Japanese Patent Publication No. 2000-515006 and Brenner S. et al. 23 Nature Biotechnology 2000 vol. 18, p630-634), etc. The sequence may be determined by As the base sequence of the low molecular weight RNA whose sequence has been determined, it is preferable to select a sequence that matches the genomic sequence using the genome database of the organism from which it was derived. That is, it is preferable to exclude low molecular RNA sequences that do not match the genomic base sequence. Furthermore, it is preferable to exclude sequences that are mRNA, tRNA, and rRNA in comparison with the database. Since noise can be removed by performing the above examination, the efficiency of selection performed later can be improved. The database is not particularly limited, and for example, a UCSC genome database, an NCBI Refseq database, the European ribosomal RNA database, and the Genomic tRNA database can be used.

  Next, based on the determined low molecular RNA base sequence, the genomic sequences before and after the low molecular RNA base sequence are obtained from the genomic information of the living organism. It is conceivable that the sequence of the loop region of the stem loop structure is included in this low molecular RNA base sequence and the sequences before and after that. The length of the low-molecular RNA base sequence for selecting the loop sequence and the sequence before and after it are not particularly limited, but for example, 20 to 500 bases, 40 to 300 bases, 60 to 260 bases, 80 to 220 bases, 110 to 200 bases A base is preferred. From the genomic sequences before and after this low molecular RNA base sequence, (A) the free energy is calculated by the Zuker method, and the region of the region where the free energy is low, preferably the region where the free energy is the lowest, is selected. That is, in the genomic sequences before and after the low molecular RNA base sequence, free energy is calculated for each region where a region of a certain length (for example, a region of 110 bases) is shifted by one base from the end, for example, Select the region with the lowest free energy. The lowest free energy is −25.0 kcal / mol or less, preferably −30.0 kcal / mol or less, more preferably −85.0 to −30.0 kcal / mol or less. In addition, the region that has the lowest free energy and is expected to form a stem-loop structure is selected. Further, (B) a sequence in which the base sequence of the low molecular RNA is present in the stem region of the stem loop structure is selected. For these selections, for example, RNA fold and mfold of Vienna RNA Package can be used. That is, a genomic sequence including a specific sequence and a complementary sequence in the opposite direction is expected to form a stem-loop structure, and a sequence sandwiched between the two sequences is a loop sequence.

  Next, an shRNA consisting of a loop sequence selected from the genome sequence and a stem region containing a target gene for gene repression or a partial sequence thereof and a complementary sequence in the reverse direction is constructed. This shRNA may be chemically synthesized, or DNA that can be transcribed by RNA polymerase may be prepared and generated by an RNA transcription system.

  By confirming the effect of shRNA constructed as described above on gene repression, the effect of the loop region on gene repression can be evaluated. The loop sequence of shRNA that causes strong gene suppression is a loop sequence useful for gene suppression. The method for confirming the effect of gene suppression is not particularly limited. For example, there are a method of directly introducing shRNA into a cell, a method of introducing a DNA construct into which shRNA is transcribed in the cell, and an intracellular target. It can be confirmed by detecting the transcription and translation of the gene. Many vectors, hosts, and kits for confirming gene suppression are commercially available. These kits can be suitably used in the screening method of the present invention. Although not particularly limited, for example, shRNA containing a loop sequence obtained by the screening method of the present invention and a sequence having a part of a nucleic acid encoding the fluorescent protein as a stem region is applied to a cell expressing the fluorescent protein. Introducing and quantifying fluorescent protein in cells can evaluate the effect on gene suppression in the loop region.

  The shRNA containing the loop sequence selected by the screening method of the present invention can effectively suppress gene expression by the mechanism of siRNA or miRNA, and is useful for gene function analysis, gene therapy, and the like.

2. Nucleic acid, vector, kit containing shRNA loop sequence useful for gene suppression The nucleic acid containing the shRNA loop sequence useful for gene suppression of the present invention is the above-mentioned 1. A nucleic acid containing a loop sequence obtained by the described screening method. The length of the loop sequence of the shRNA of the present invention is 2 to 200 bases, preferably 4 to 50 bases, particularly preferably 8 to 20 bases. Examples of such base sequences include loop sequences described in SEQ ID NOs: 12 to 20 in the Sequence Listing. Moreover, in the sequence | arrangement in any one of sequence number 12-20 of a sequence table, 1 or more, Preferably 1 or more, Especially preferably, 1 or several, More preferably, the lack of 1-10 bases Examples thereof include those represented by sequences having at least one of deletion, addition, insertion or substitution, and useful for gene suppression. Here, as a sequence having at least one of deletion, addition, insertion or substitution of one or more bases in the sequence described in any one of SEQ ID NOs: 12 to 20 in the sequence listing, for example, SEQ ID NO: 12-20 A nucleic acid sequence having 50% or more homology to any nucleotide, preferably a nucleic acid sequence having 70% or more homology to the nucleotide, particularly preferably a nucleic acid sequence having 90% or more homology to the nucleotide Illustrated. A person skilled in the art can easily prepare and order shRNA containing a loop sequence based on the nucleic acid sequence of the present invention, and easily introduce the prepared shRNA into a cell expressing a target gene or the like for gene repression. Since the effect can be confirmed, a nucleic acid in which at least one of deletion, addition, insertion or substitution of one or more bases has been performed based on the nucleic acid of the present invention should be construed to be included in the present invention.

  Furthermore, the nucleic acid of the present invention includes a nucleic acid that can hybridize to the above-described nucleic acid under stringent conditions and is useful for gene suppression. The stringent conditions are described in 1989, Cold Spring Harbor Laboratory, J. Am. Examples of the conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning: A Laboratory Manual 2nd ed.) And the like are exemplified. Specifically, for example, conditions of incubating with a probe at 65 ° C. for 12 to 20 hours in 6 × SSC containing 0.5% SDS, 5 × Denharz solution, and 0.01% denatured salmon sperm DNA can be mentioned. The nucleic acid hybridized to the probe can be detected after removing the non-specifically bound probe by washing at 37 ° C. in 0.1 × SSC containing 0.5% SDS, for example.

  RNA in which a base sequence of a target gene or a part of the base sequence and a complementary sequence thereof are arranged in opposite directions across the nucleic acid of the present invention can effectively suppress the target gene. It is useful for gene suppression using dsRNA. The length of the stem region is not particularly limited as long as it is a length that generates dsRNA that can be used in gene suppression, and is, for example, 10 to 200 bases, preferably 14 to 30 bases, and particularly preferably 19 to 24 bases. .

  In addition, a vector useful for gene suppression in which the nucleic acid of the present invention is inserted into an appropriate expression vector is also included in the present invention. As long as the shRNA containing the loop sequence of the present invention is transcribed, the vector of the present invention, that is, a transcriptional regulatory sequence such as a promoter, 3′UTR, 5′UTR, terminator, etc. Any vector having a transcription termination sequence such as a poly A signal may be used. For example, the pSINsi-hU6 and other pSINsi vector series (Takara Bio), pBAsi vector series (Takara Bio), piGENE vector (iGENE), and pSIREN vector (Clontech) downstream of the loop sequence of the present invention. The inserted vector is useful because it immediately becomes a shRNA expression vector when a target gene or a partial sequence thereof is incorporated. In that case, the vector may be constructed so that the target gene or a partial sequence thereof is an inverted repeat sequence so as to form a shRNA stem region, and the loop sequence is sandwiched.

  Furthermore, a kit containing at least one nucleic acid containing the shRNA loop sequence of the present invention or a vector of the present invention is also included in the present invention. The kit of the present invention may further contain a host organism, cell, tissue, organ, and may contain a vector or nucleic acid construct for transcription and expression of the target gene in these hosts. Further, a transformation reagent for introducing the nucleic acid into the host may be included.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.
Among the operations described in this specification, the basic operations are described in 2001, issued by Cold Spring Harbor Laboratory. Edited by T. Maniatis et al., Molecular Cloning: The method described in A Laboratory Manual 3rd edition (Molecular Cloning: A Laboratory Manual 3rd ed.).

  Furthermore, E. coli TOP10 (manufactured by Invitrogen) was used as a host in the following plasmid construction using E. coli unless otherwise specified. The transformed E. coli is LB medium (tryptone 1%, yeast extract 0.5%, NaCl 0.5%, pH 7.0) containing 30 μg / ml chloramphenicol, or 1.5% in the above medium. The cells were aerobically cultured at 37 ° C. using LB-chloramphenicol plates solidified by adding% agar.

Example 1 Sample Preparation (1) RNA Extraction Human fetal kidney-derived 293T / 17 cells (ATCC CRL-11268) were present in DMEM medium containing 10% fetal bovine serum (FBS; manufactured by Gibco) in the presence of 5% CO _2. The cells were cultured at 37 ° C. for 7 days. Cells were collected, and total RNA was extracted with Trizol reagent (Gibco).

(2) Separation of low molecular weight 1 mg of 293T / 17 cell-derived total RNA was concentrated with Microcon 30 (Millipore). The concentrated small RNA was electrophoresed on 15% TBE-urea gel (Invtrogen). After electrophoresis, about 21 to 27 bases were excised and low molecular RNA was separated from the gel.

(3) Preparation of small RNA tag library After digesting the tag vector pMBS1 described in US Publication No. 2004/0002104 with BamHI and BbsI (both made by New England Biolab (NEB)), bovine intestinal alkaline phosphatase ( CIAP (manufactured by Takara Bio Inc.) was used for dephosphorylation.

E. coli C75 alkaline phosphatase (BAP C75, manufactured by Takara Bio Inc.) was allowed to act on 30 ng of low-molecular RNA derived from 293T / 17 cells to perform dephosphorylation. Subsequently, the synthetic RNA / DNA chimera oligo shown in SEQ ID NO: 1 in the sequence listing in which 6 bases on the 5 ′ side are RNA at the 3 ′ end of this low molecular RNA and the others are DNA is T4 RNA Ligase (Takara Bio). ). Thereafter, electrophoresis was performed with 15% TBE-urea gel (manufactured by Invtrogen). After electrophoresis, the target product was excised and separated from the gel.
This synthetic oligo-linked small RNA was subjected to T4 polynucleotide kinase (manufactured by Takara Bio Inc.) to phosphorylate the 5 ′ end. Thereafter, a synthetic DNA / RNA chimera oligo represented by SEQ ID NO: 2 in the sequence listing in which 6 bases on the 3 ′ side were RNA at the 5 ′ end and DNA was the other was ligated with T4 RNA Ligase (Takara Bio Inc.). Furthermore, electrophoresis was performed with 15% TBE-urea gel (Invtrogen). After electrophoresis, the target product was removed from the gel.

  M-MLV RTase (manufactured by Takara Bio Inc.) using as a template low molecular RNA with synthetic oligos linked to both ends and using the primer shown in SEQ ID NO: 3 in the sequence listing as a substrate for dCTP, dATP, dGTP, and dTTP. A reverse transcription reaction was performed. Using this DNA as a template, PCR was performed using the FAM-labeled oligonucleotide represented by SEQ ID NO: 4 in the Sequence Listing and the FAM-labeled oligonucleotide represented by SEQ ID NO: 5 in the Sequence Listing as primers. The PCR reaction was performed using Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) with 5'-methylated-dCTP, dATP, dGTP, and dTTP as substrates. This PCR product was purified by phenol treatment, chloroform treatment and ethanol precipitation.

The purified PCR product was digested with SfaNI (NEB) to purify the DNA. This DNA was electrophoresed on a 15% acrylamide gel, the band of the target DNA fragment was cut out, and the DNA fragment was extracted from the gel.
The cDNA fragment obtained by the above method and the linearized pMBS1 described above were ligated with T4 DNA Ligase (manufactured by Takara Bio Inc.), and E. coli TOP10 was obtained by electroporation using the obtained recombinant plasmid. Transformed. A part of the transformant is inoculated on an LB-chloramphenicol plate, the number of clones is calculated from the number of colonies generated, and the remaining transformant is inoculated on an LB medium containing LB-chloramphenicol. Plasmid DNA was purified from a culture with a clone number of 640,000 using QIAGEN Plasmid Midi Kit (Qiagen) to obtain a tag library.

Example 2 Preparation of Microbeads PCR was performed using the above tag library as a template. PCR uses 5′-methylated-dCTP, dATP, dGTP, and dTTP as substrates, and the primer is the oligonucleotide shown in SEQ ID NO: 6 in the sequence listing, the FAM-labeled oligonucleotide shown in SEQ ID NO: 7 and the sequence in the sequence listing A biotinylated oligonucleotide represented by No. 7 was mixed at a ratio of 9: 1 and reacted with Ex Taq Hot Start Version (manufactured by Takara Bio Inc.). After purifying the PCR product, digestion with restriction enzyme PacI (manufactured by NEB), and further, T4 DNA polymerase (manufactured by NEB) was allowed to act in the presence of dGTP, and the tag portion was single-stranded, DNA was purified.

30 μg of a single-stranded tagged target DNA fragment and 7.2 × 10 ⁷ anti-tag-bound microbeads (manufactured by Solexa) were mixed, and 100 μl of 500 mM NaCl, 11.6 mM sodium phosphate, 0.01% Hybridization was performed at 69 ° C. for 3 days in Tween 20, 3.5% dextran sulfate. Two reactions were performed. The microbeads were washed with 10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.01% Tween 20, and two microbeads were combined into one.

  By allowing T4 DNA ligase to act on the washed microbeads, a covalent bond was formed between the target DNA fragment and the anti-tag. Then, it was made to bind to 600 μg Dynabead M-280 streptavidin (magnetic streptavidin beads, manufactured by Dynal) for 30 minutes at 25 ° C., and left to stand for 1 minute in MPC (manufactured by Dynal), and then the supernatant was removed. Repeated washing operation of resuspending in 1 ml of 10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.01% Tween 20 and allowing to stand on MPC (manufactured by Dynal), and then removing the supernatant. Only the microbeads carrying the tagged target DNA fragment were isolated.

  The separated microbeads were digested with MboI (manufactured by Takara Bio Inc.) and cut out from Dynabeads M-280 streptavidin. Further, Klenow Fragment was allowed to act on the microbeads in the presence of dGTP, and then divided into two equal parts, and two types of adapter DNAs were ligated using T4 DNA ligase. The adapter DNA is obtained by annealing the oligonucleotide shown in SEQ ID NO: 8 in the sequence listing and the oligonucleotide shown in SEQ ID NO: 9 in the sequence listing, and the oligonucleotide shown in SEQ ID NO: 10 in the sequence listing and the sequence listing. The oligonucleotide shown in SEQ ID NO: 11 is annealed.

Example 3 Extraction of MicroRNA Candidate Sequences by MPSS Analysis (1) MPSS Analysis The above microbeads were obtained from Japanese Patent Publication No. 2000-515006 and Brenner S. et al. 23 others Nature Biotechnology 2000 vol. 18, using the technique disclosed in p630-634, the sequence of 22 base sequences of the target DNA immobilized on the microbeads was read, and the number of the same sequences was calculated. Next, the calculated numbers were summed up, and the number of individual arrays was divided by the total number and multiplied by 1 million to calculate the number of individual arrays per million.

(2) Extraction of novel microRNA candidate sequences from MPSS data The homology search of the 4737 sequences obtained by the above MPSS analysis was performed on the UCSC genome database (http://genome.ucsc.edu) to match the genome sequence 906 sequences were extracted. Subsequently, the Sanger micro RNA database (http://microrna.sanger.ac.uk/), NCBI Refseq database (http://www.ncbi.nlm.nih.gov/RefSeq/), the European ribosomal RNA database (http : //www.psb.ugent.be/rRNA/), a homology search to the Genomic tRNA database (http://lowelab.ucsc.edu/GtRNAdb/) to extract 243 sequences that do not match any database did. Thereafter, both ends of the sequence are extended by 88 bases from the genomic sequence information that matches the extracted sequence, a 198 base sequence is obtained, and the 110 base sequence is shifted from the end of this sequence by one base at a time. 110 base sequences differing by one base were prepared. The secondary structure prediction of the 110 base sequence is performed using the RNA fold of the Vienna RNA Package (http://www.tbi.univie.ac.at/~ivo/RNA/), and the free energy is minimal. In the 22-base sequence obtained by MPSS analysis in the stem portion, 16 stems or more complementary to the complementary strand side were extracted, and 42 novel microRNA candidates were obtained. . Nine kinds of sequences were randomly selected from the obtained novel microRNA candidates, and loop sequences were obtained from the stem loop sequences (shown in SEQ ID NOs: 12 to 20 in the Sequence Listing). The nine selected free energies were -84.1 to -32.7 kcal / mol.

Example 4 Preparation of shRNA Expression Vector Having Various Loop Sequences shRNA expression vectors for green fluorescent protein rsGFP target sequence A (GGAGTTGTCCCAATTTCTG) (SEQ ID NO: 27) and target sequence B (GACACGTGCTGAAGTCAAG) (SEQ ID NO: 28) were obtained by the following procedure. Produced. Expression of stem-loop structure shRNA having the loop sequence of SEQ ID NO: 12 to 20 with respect to the target sequence (that is, RNA in which the sequence of SEQ ID NO: 27 or 28 and the complementary sequence in the opposite direction are connected to both ends of either loop sequence) A synthetic oligo DNA was inserted into the downstream of the hU6 promoter of the expression vector pSINsi-hU6 (manufactured by Takara Bio Inc.) in accordance with the procedure of the product instructions to construct a plasmid vector. As a control, a vector expressing the loop of the sequence shown in SEQ ID NO: 21 (Brummelkamp et al. Science. 2002 296: 550-553.), Which had been used for the loop structure of the shRNA expression vector so far, was prepared. As a negative control, a vector into which nothing was inserted was prepared. The obtained various vectors were transformed into E. coli JM109, and the plasmid DNA was purified using QIAGEN Plasmid Midi Kit (manufactured by Qiagen) and used as transfection DNA.

Example 5 Preparation of shRNA-expressing retroviral vector having various loop sequences The plasmid vector prepared in Example 4 was transfected into 293T / 17 cells using Retrovirus Packaging Kit Ampho (manufactured by Takara Bio Inc.) according to the product protocol. Various amphotropic virus supernatants were obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and stored in a -80 ° C. ultra-low temperature freezer until use.

Example 6 Confirmation of Gene Suppression Effect by Retroviral Vector HT1080 cells (ATCC CCL-121) and K562 cells (ATCC CCL-243) stably expressing the target gene rsGFP were prepared by the following procedure.
The rsGFP expression vector pQBI25 (manufactured by Qbiogene) was cleaved with restriction enzymes NheI and NotI to obtain a GFP gene fragment of 775 bp. Next, pQBI polII (manufactured by Qbiogene) is cleaved with restriction enzymes NheI and NotI to remove the rsGFP-NeoR fusion gene, and the previously obtained 775 bp rsGFP gene fragment is inserted, and the rsGFP gene is expressed under the control of the polII promoter. The vector pQBI polII (neo-) was obtained. pQBI pol II (neo-) was digested with the restriction enzyme Xho I to obtain a DNA fragment containing a GFP expression unit under the control of the pol II promoter, and its ends were blunted using a DNA blunting kit (manufactured by Takara Bio Inc.). After blunting the end of the vector fragment 4.58 kbp obtained by digesting the retroviral vector plasmid pDON-AI (Takara Bio) with restriction enzymes XhoI and SphI using a DNA blunting kit (Takara Bio) Then, dephosphorylation was performed using alkaline phosphatase (manufactured by Takara Bio Inc.). A DNA fragment containing an rsGFP expression unit under the control of the previously smoothed pol II promoter was inserted into this smoothed vector using DNA Ligation Kit (manufactured by Takara Bio Inc.) to obtain an rsGFP-expressing recombinant retroviral vector pDOG-pol II. It was. Moreover, the smoothed GFP gene was inserted into the smoothed vector to obtain an rsGFP-expressing recombinant retroviral vector pDOG.

These plasmid vectors were transfected into retrovirus preparation cells G3T-hi (manufactured by TAKARA BIO INC.) Using Retrovirus Packaging Kit Ampho according to the product protocol to obtain various amphotropic virus supernatants, and 0.45 μm filters (Milex HV, manufactured by Millipore) and stored in an -80 ° C ultra-low temperature freezer until use.
HT1080 cells were seeded on a 6-well tissue culture plate (Iwaki Glass Co., Ltd.) at 5 × 10 ⁴ cells per well, and in 10% fetal bovine serum (FBS) -containing DMEM medium at 37 ° C. in the presence of 5% CO _2. For 24 hours.

The amphotropic DOG virus solution was serially diluted, and infection was carried out in the presence of 8 μg / ml of polybrene (hexadimethrine bromide; Sigma). After culturing for 3 days, GFP expression was analyzed by a flow cytometer (FACS Vantage, manufactured by Becton Dickinson), GFP positive cells were collected from a sample with an introduction efficiency of 20% or less by sorting, cultured, and cultured for stable GFP expression. Cells were HT1080-GFP.
K562 cells were cultured at 37 ° C. in the presence of 5% CO _{2 in} RPMI 1640 medium containing 10% fetal bovine serum (FBS).

  The amphotropic DOG-pol II virus solution was serially diluted, and infection was performed by a standard method using RetroNectin (registered trademark, manufactured by Takara Bio Inc.). The cells were cultured for 3 days after infection, and GFP expression was analyzed with a flow cytometer. GFP positive cells were collected from a sample with an introduction efficiency of 20% or less by sorting and cultured to obtain GFP stably expressing cells K562-GFP.

HT1080-GFP and K562-GFP thus prepared were infected with the retroviral vector prepared in Example 5. HT1080-GFP was diluted 10-fold and 100-fold with a viral vector and infected in the presence of 8 μg / ml polybrene. K562-GFP was infected by a standard method using retronectin after diluting the viral vector with a stock solution and 10-fold. After 24 hours from the infection, HT1080-GFP was replaced with a growth medium containing 500 μg / ml of G418 (Geneticin; manufactured by Invitrogen), and K562-GFP was replaced with a growth medium containing 1000 μg / ml of G418 and selected for 2 weeks. Culture was performed.
After selective culture for 2 weeks to obtain cells stably expressing siRNA, the cells were analyzed with a flow cytometer, and the fluorescence intensity of GFP was calculated. The gene suppression effect was evaluated by calculating the ratio of the fluorescence intensity in each experimental group to the fluorescence intensity of the control experimental group. The result of HT1080-GFP at the target sequence A is shown in FIG. 1, and the result of K562-GFP at the target sequence A is shown in FIG. The result of HT1080-GFP in the target sequence B is shown in FIG. 3, and the result of K562-GFP in the target sequence B is shown in FIG. In the figure, the vertical axis indicates the relative value of GFP fluorescence intensity when the negative control (Negative control) is 100 (Relative GFP mean (%)). The numbers on the horizontal axis indicate the sequence numbers. As shown in the figure, the gene suppression effect of the loop sequences shown in SEQ ID NOs: 12, 13, 14, 15, 16, 18, 19, and 20 is higher than that of the loop sequence shown in SEQ ID NO: 21. Indicated.

Example 7 Preparation of shRNA Expression Vector Having Various Loop Sequences An shRNA expression vector for human integrin α4 target sequence (GAGTGTTGGTTACACAA) (SEQ ID NO: 29) was prepared by the following procedure. SEQ ID NOs: 13 and 19 used in Example 4 for the target sequence, and the stem-loop sequence of microRNA-30, which has recently been used with high siRNA effect (Boden et al. Nucleic Acids Research 2004 32 (3): 1154-1158), a synthetic oligo DNA for expressing shRNA having the loop sequence of SEQ ID NO: 22 (that is, RNA having SEQ ID NO: 29 and a complementary sequence in the opposite direction linked to either end of the loop sequence) A plasmid vector was constructed by inserting it into the downstream of the hU6 promoter of the expression vector pSINsi-hU6 (manufactured by Takara Bio Inc.) according to the procedure described in the product instructions. As a control, a vector expressing the loop of the sequence shown in SEQ ID NO: 21 (Brummelkamp et al. Science. 2002 296: 550-553.), Which had been used for the loop structure of the shRNA expression vector so far, was prepared. As a negative control, a plasmid vector having an rsGFP target sequence A shown in SEQ ID NO: 27 and a loop of the sequence shown in SEQ ID NO: 21 prepared in Example 4 was used. Escherichia coli JM109 was transformed with the various vectors obtained, and the plasmid DNA was purified using QIAGEN Plasmid Midi Kit (manufactured by Qiagen) and used as transfection DNA.

Example 8 Preparation of shRNA-expressing retroviral vector having various loop sequences The plasmid vector prepared in Example 7 was added to retrovirus preparation cell G3T-hi (manufactured by Takara Bio Inc.) and Retrovirus Packaging Kit Ampho (manufactured by Takara Bio Inc.). Was used to obtain various amphotropic virus supernatants, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and stored in a -80 ° C. ultra-low temperature freezer until use. . Each virus was prepared in two cases.

Example 9 Retrovirus vector titer measurement The retrovirus vector prepared in Example 8 was serially diluted and infected with HT1080 cells (ATCC CCL-121) in the presence of 8 μg / ml of polybrene. After 24 hours from the infection, G418 (Geneticin; manufactured by Invitrogen) was replaced with a growth medium containing 500 μg / ml, and selective culture was performed for 2 weeks. The retrovirus vector titer was calculated from the number of colonies formed.

Example 10 Confirmation of Gene Suppression Effect by Retroviral Vector A retroviral vector prepared in Example 8 for HL60 (ATCC CCL-240) cells and titered in Example 9 was used as a standard using retronectin at MOI2. Infection was performed in a typical way. After 24 hours from the infection, G418 was replaced with a growth medium containing 1000 μg / ml, and selective culture was performed for 2 weeks.

  After selective culturing for 2 weeks, cells stably expressing siRNA were collected, and total RNA was extracted and treated with DNAseI with QIAGEN RNeasy Mini Kit (Qiagen). The extracted total RNA was subjected to reverse transcription reaction with Reverse Transscriptase M-MLV (manufactured by Takara Bio Inc.) using a random primer (6mer), SYBR Premix Ex Taq (manufactured by Takara Bio Inc.) and SEQ ID NOS: 23 and 24. Real-time PCR was performed using the integrin α4 amplification primer, and the relative value of the integrin α4 gene expression level was calculated. The total RNA amount was corrected using the GAPDH gene amplification primers of SEQ ID NOs: 25 and 26.

  The gene suppression effect was evaluated by calculating the ratio of the expression relative value in each experimental group to the integrin α4 expression relative value in the control experimental group. The results are shown in FIG. In the figure, the vertical axis indicates the integrin α4 expression level as a relative value when the negative control (Negative control) is 100 (Relative integrin α4 mean (%)). The numbers on the horizontal axis indicate the sequence numbers. As shown in FIG. 5, compared with the loop sequence shown in SEQ ID NO: 21 and the loop sequence of microRNA-30 shown in SEQ ID NO: 22, the gene suppression effect of the loop sequences shown in SEQ ID NOs: 13 and 19 is higher. It was shown to be expensive. Furthermore, the order of the gene suppressive effect of the loop sequences compared this time was consistent with the results of the gene suppressive effect on GFP performed in Examples 4, 5, and 6.

  According to the present invention, nucleic acids encoding shRNA loop regions useful for gene suppression can be easily screened. Furthermore, a nucleic acid encoding a shRNA loop region useful for gene suppression and having a gene suppression effect larger than that of a conventionally used shRNA loop sequence, a vector containing the nucleic acid, and a kit containing the nucleic acid or vector are provided. It becomes possible to do.

SEQ ID NO: 1: Synthetic chimera oligonucleotide. "Nucleotide 1 to 6 are ribonucleotide-other nucleotides are deoxyribonucleotides
SEQ ID NO: 2: Synthetic chimera oligonucleotide. "Nucleotide 17 to 22 are ribonucleotide-other nucleotides are deoxyribonucleotides
SEQ ID NO: 3: Synthetic primer for revearse transcription.
SEQ ID NO: 4: Synthetic primer.
SEQ ID NO: 5: Synthetic primer.
SEQ ID NO: 6: Synthetic primer.
SEQ ID NO: 7: Synthetic primer.
SEQ ID NO: 8: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO: 9: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO: 10: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO: 11: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO: 12: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 13: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 14: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 15: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 16: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 17: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 18: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 19: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 20: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 21: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 22: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 23: Synthetic primer for amplification of integrin alpha 4 gene
SEQ ID NO: 24: Synthetic primer for amplification of integrin alpha 4 gene
SEQ ID NO: 25: Synthetic primer for amplification of GAPDH gene
SEQ ID NO: 26: Synthetic primer for amplification of GAPDH gene
SEQ ID NO: 27: Green fluorescence protein rsGFP target sequence A
SEQ ID NO: 28: Green fluorescence protein rsGFP target sequence B
SEQ ID NO: 29: Human integrin alpha 4 target sequence

Claims (4)

a loop sequences for shRNA, nucleic acid comprising a base sequence described in SEQ ID NO 19 of the Sequence Listing.   A vector for expressing shRNA comprising the nucleic acid according to claim 1.   A kit comprising the nucleic acid according to claim 1 or the vector according to claim 2.   ShRNA comprising a nucleic acid whose loop sequence consists of the base sequence set forth in SEQ ID NO: 19 in the Sequence Listing.

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