WO2005017144A1 - dsRNA分解およびRNA合成方法 - Google Patents
dsRNA分解およびRNA合成方法 Download PDFInfo
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- WO2005017144A1 WO2005017144A1 PCT/JP2004/011480 JP2004011480W WO2005017144A1 WO 2005017144 A1 WO2005017144 A1 WO 2005017144A1 JP 2004011480 W JP2004011480 W JP 2004011480W WO 2005017144 A1 WO2005017144 A1 WO 2005017144A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Definitions
- the present invention relates to a protein having an activity of producing a dsRNA of a specific length, a protein having a dsRNA degrading activity, a dsRNA obtained by combining the protein with a protein having a nucleic acid binding activity, for example, a protein having an RNA binding activity.
- the present invention relates to a method for efficiently decomposing RNA and a method for efficiently synthesizing RNA by combining a protein having nucleic acid binding activity and a protein having RNA synthesis activity.
- RNA interference is a phenomenon in which mRNA is degraded by dsRNA in a sequence-specific manner, resulting in suppression of gene expression.
- the inception that dsRNA was able to silence genes came from studies using antisense in nematodes.
- Guo and Kemphues conducted an experiment in which a gene called par-1 was suppressed with antisense RNA.
- Addition of antisense RNA suppressed the expression of par ⁇ l as expected
- the sense RNA used as a control also suppressed the expression of par-1 and the phenotype of the par-1 mutant showed that.
- Non-Patent Document 2 When synthesizing antisense RNA and sense RNA using RNA polymerase, respectively, slightly nonspecific reverse RNAs are generated. The dsRNA generated by the contamination is the main component of gene silencing, antisense RNA and sense RNA cannot suppress gene expression, and dsRNA that anneals antisense RNA and sense RNA is effective. It was found that gene expression can be suppressed efficiently. (For example, Non-Patent Document 2)
- RNA interference an enzyme called Dicer generates small molecule RNA (sRNA: short interfering RNA) from dsRNA.
- sRNA small molecule RNA
- dsRNA short interfering RNA
- Non-Patent Document 4 RNA induced silencing com plex
- Non-Patent Document 5 human-derived Dicer
- Non-Patent Document 6 a recombinant Dicer
- dsRNA having a chain length of about 21 nucleotides synthesized using RNA polymerase is used as it is as an siRNA (for example, Non-Patent Document 7). Therefore, if there is a method that can efficiently produce RNA, it can be used for the above method.
- Non-patent literature l Guo S. et al. Cell 1995 vol. 81, p611_620
- Non-patent document 2 Fire A. et al. 5 Nature 1998 vol. 39, ⁇ 806 ⁇ 811
- Non-patent document 3 Bernstein ⁇ . Et al. 3 people Nature 2001 vol. 409, p363_366
- Non-patent document 4 Tabara H. et al. 3 Name Cell 2002 vol.109, p861-871
- Non-Patent Document 5 Zhang H. et al. 4 The EMBO Journal 2002 vol.21, No
- Non-Patent Document 6 Myers J.W. and 3 others Nature biotechnology 2003 vol. 21, p324-328
- Non-patent Document 7 Donze O. et al. 1 Nucleic Acids Research 2002 vol. 30, No. 10, e46 Disclosure of the invention
- the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, analyzed the functional domain of Dicer, and found that it has the activity of acting on long-chain dsRNA to generate dsRNA of a specific length. A protein having dsRNA degradation activity was found.
- a dsRNA having a specific length can be efficiently prepared by allowing a dsRNA to act on a dsRNA in the presence of a protein having a nucleic acid binding activity, for example, a protein having an RNA binding activity.
- a protein having the nucleic acid binding activity improves the efficiency of an RNA synthesis reaction represented by dsRNA synthesis, and completed the present invention.
- a first invention of the present invention is a dsRNA degrading activity, which is a protein having a dsRNA degrading activity, wherein the protein has an activity of acting on dsRNA to generate a dsRNA of a specific length.
- the protein having dsRNA-degrading activity preferably has a functional domain of Dicer, and for example, preferably comprises RNaseIIIa, b and a dsRNA-binding domain. In addition, even if it contains a PAZ domain. Further, by using the protein of the first invention of the present invention, dsRNA of a specific length can generate dsRNA of about 15 to 30 base pairs. Further, as the protein of the first invention of the present invention, the protein having dsRNA degrading activity is encoded by the amino acid sequence of SEQ ID NO: 4 or 17 in the sequence listing or the nucleotide sequence of SEQ ID NO: 3 or 16 in the sequence listing.
- a protein comprising an amino acid sequence is exemplified.
- it may be a protein consisting of an amino acid sequence represented by SEQ ID NO: 4 or 17 in the sequence listing, in which one or more amino acids have been substituted, deleted, inserted or added.
- the protein having the dsRNA-degrading activity of the first invention of the present invention has a codon suitable for expression in a host. It can be efficiently produced by conversion or by using a host reinforced for rare codons.
- the protein can be expressed using a cold-inducible vector.
- the protein of the first invention of the present invention can be contained in a kit as a component.
- a second invention of the present invention is characterized in that a dsRNA having a dsRNA-degrading activity is allowed to act on dsRNA in the presence of a protein having a nucleic acid binding activity to produce a dsRNA of a specific length.
- the present invention relates to a method for degrading dsRNA.
- the protein having nucleic acid binding activity and the protein having dsRNA degradation activity may be fusion proteins.
- the protein having nucleic acid binding activity may be a protein having RNA binding activity.
- the protein having the RNA binding activity may be a cold shock protein or may be derived from a thermophilic bacterium or a thermostable bacterium.
- cold shock protein B derived from Thermotoga maritima is exemplified.
- a dsRNA having a specific length of about 15 to 30 base pairs can be produced.
- the protein having the dsRNA degrading activity may be the protein of the first invention of the present invention, or may be a natural Dicer or a functional equivalent thereof. .
- the third invention of the present invention relates to a method for synthesizing RNA, which comprises performing an RNA synthesis reaction using a protein having RNA synthesis activity in the presence of a protein having nucleic acid binding activity.
- the protein having nucleic acid binding activity and the protein having RNA synthesis activity may be fusion proteins.
- the protein having the nucleic acid binding activity may be a cold shock protein, or may be derived from a thermophilic bacterium or a thermostable bacterium.
- cold shock protein B derived from Thermotoga maritima is exemplified.
- the protein having the activity of synthesizing RNA may be a DNA-dependent RNA polymerase.
- a fourth invention of the present invention is a composition for use in the method of the second invention of the present invention.
- a protein having a nucleic acid binding activity and a protein having a dsRNA degrading activity is a composition for use in the method of the second invention of the present invention.
- a fifth invention of the present invention is a kit for use in the method of the second invention of the present invention.
- a kit comprising a protein having nucleic acid binding activity and a protein having dsRNA degrading activity.
- a sixth invention of the present invention is a composition for use in the method of the third invention of the present invention, which comprises a protein having a nucleic acid binding activity and a protein having an RNA synthesis activity.
- the composition features.
- a seventh invention of the present invention is a kit for use in the third method of the present invention, which comprises a protein having a nucleic acid binding activity and a protein having an RNA synthesis activity. About.
- a protein having a dsRNA degrading activity capable of preparing dsRNA of a specific length. Further, according to the present invention, dsRNA of a specific length that can be used for RNA interference or the like can be efficiently produced.
- Dicer refers to a protein having a function of processing long dsRNA into siRNA at an early stage of RNAi.
- Examples of the natural Dicer include, but are not limited to, those composed of an ATP binding domain, an RNA helicase domain, a PAZ domain of unknown function, RNasellla and b domains, and a dsRNA binding domain from the N-terminal side.
- the functional domain of Dicer refers to a site encoding a region involved in an activity capable of producing a dsRNA of a specific length by acting on a long dsRNA.
- the functional domain is not particularly limited, and examples thereof include RNasellla, b domain and dsRNA binding domain.
- the RNaseIIIa and b domains specifically act on double-stranded RNA, as described in Zhang H. et al., Four, The EMBO Journal 2002 vol. 21, No. 21, p5875-5885, and 5 ′
- the dsRNA binding domain may be a site encoding an activity that specifically binds to double-stranded RNA.
- dsRNA refers to RNA that forms a double-stranded structure of an mRNA to be subjected to RNA interference and an RNA having a complementary nucleotide sequence to the mRNA.
- examples of application of a product obtained by a degradation reaction of dsRNA include siRNA mainly shown below.
- the dsRNA of a specific length is not particularly limited, but refers to, for example, a dsRNA of a specific length in a range of about 100 base pairs.
- the dsRNA may be of a specific length in the range of about 1530 base pairs, particularly of a specific length in the range of 2025 base pairs.
- These dsRNAs can be used as siRNA.
- protein having nucleic acid binding activity refers to a protein having an activity of binding to single-stranded or double-stranded DNA or RNA.
- proteins having a function of resolving the secondary structure of a nucleic acid are preferable, and examples thereof include DNA helicase, RNA helicase and functional equivalents thereof.
- cold-inducible vector refers to a vector having a promoter that can function at a low temperature, such as a p Cold vector described in WO 99/27117. .
- cold shock protein is a general term for proteins that are expressed by being stimulated by a decrease in temperature under a condition where the temperature is lower than the original growth condition.
- the term "completely decompose a long dsRNA serving as a substrate” refers to decomposing a long dsRNA serving as an uncleaved substrate after a decomposition reaction to such an extent that it is not confirmed by electrophoresis. .
- the PAZ domain is a domain existing between the RNA helicase domain of Dicer and the RNasellla and b domains.
- it is a region of amino acid numbers 898-1064 (base numbers 269 2-3192 of SEQ ID NO: 2) in the amino acid sequence of Dicer of human origin described in SEQ ID NO: 1 in the sequence listing.
- the rare codon means a codon that is rarely used.
- One amino acid Depending on the species that is defined by multiple codons, there is a bias in the frequency of use between these multiple codons.
- tRNA transfer RNA
- the protein having dsRNA-degrading activity of the present invention can act on dsRNA to produce dsRNA of a specific length.
- the protein having the dsRNA-degrading activity is not particularly limited as long as it can generate a dsRNA of a specific length from a long-chain dsRNA.
- a protein having a functional domain of Dicer is exemplified.
- the functional domain of the Dicer may be a protein comprising RNaseIIIa, b and dsRNA binding domains.
- the origin of the protein does not matter as long as it can generate dsRNA of a specific length from long dsRNA.
- RNase IIIa, b domain and dsRNA binding domain are not particularly limited, but, for example, in the case of human-derived Dicer, amino acids 1271-1924 on the N-terminal side of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing (described in SEQ ID NO: 2 in the sequence listing) Base sequence No. 3811-5772).
- a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing a protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing (Dicer mutant), or a protein consisting of the amino acid sequence shown in SEQ ID NO: 12
- examples thereof include a protein consisting of an amino acid sequence or a protein (Dicer variant) consisting of the amino acid sequence IJ encoded by the nucleotide sequence of SEQ ID NO: 13 in the sequence listing.
- Dicer is a large protein and is not suitable for recombinant production.
- the protein having dsRNA-degrading activity of the present invention is small in size and compact as compared with a native enzyme, and is useful for producing a recombinant.
- enzymes derived from higher organisms such as humans are produced as recombinants in bacterial cells such as Escherichia coli. In the case of production, it is often difficult to produce a recombinant while maintaining the same enzyme activity. Therefore, the protein having dsRNA-degrading activity of the present invention is very useful when produced in cells other than the organism from which it is derived.
- the protein of the present invention may contain a PAZ domain, but is not particularly limited.
- amino acids 898 to 1924 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing A protein having the amino acid sequence of SEQ ID NO: 17 in the Sequence Listing, or a protein having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 16 in the Sequence Listing (Dicer Mutant) or a protein (Dicer variant) consisting of the amino acid sequence represented by SEQ ID NO: 18 in the sequence listing, or an amino acid sequence encoded by the base sequence represented by SEQ ID NO: 19 in the sequence listing .
- the enzyme is possible to make more enzymatically stable by using the mutant.
- stability can be improved as compared to a mutant protein having only an RNaselll domain.
- the activity can be maintained even after more freezing and thawing, and the activity can be maintained for a long time in a solution state in a certain storage buffer.
- the protein of the present invention is not particularly limited.
- long-chain dsRNA can be degraded to generate siRNA effective for RNA interference.
- substitution, deletion, insertion or addition of one or more amino acids or bases in the above amino acid sequence or base sequence may be included in the protein having dsRNA degrading activity of the present invention. included.
- a sequence derived from an expression vector for example, an expression or translation-enhancing system IK, such as a Perfect DB sequence, etc.
- a tag sequence for purifying an expressed protein eg, For example, those having an amino acid sequence such as a His tag sequence
- a sequence for removing an additional sequence at the N-terminal side of the expressed protein eg, a Fact or Xa sequence
- proteins having The protein is not particularly limited, but has, for example, a dsRNA degrading activity having the amino acid sequence of SEQ ID NO: 12 or 18 in the sequence listing. Protein.
- the protein having a dsRNA-degrading activity of the present invention can convert a long dsRNA into a dsRNA of a specific length. That is, in the present invention, by selecting a protein having dsRNA degradation activity to be used, dsRNA having a desired specific length can be prepared.
- the dsRNA of the specific length is not particularly limited, for example, a specific length in a range of about 10-100 base pairs, preferably in a range of about 1530 base pairs, and particularly preferably in a range of about 2025 base pairs. DsRNA is exemplified.
- the protein having the dsRNA-degrading activity of the present invention for example, but not limited to, the Dicer mutant, can be stored at 4 ° C. by storing in a Tris-HCl buffer having a pH of 8.5 or more in the presence of magnesium chloride. Its activity can be stably maintained for a long period of time both in storage and storage at 20 ° C.
- the protein having dsRNA-degrading activity of the present invention is a protein having nucleic acid binding activity described in the following (2), and is not particularly limited.
- it may be in the form of a fusion protein with a protein having RNA binding activity. Is also good.
- protein having the above Dice r mutant and a nucleic acid binding activity for example, fusion proteins with proteins having RNA binding activity is exemplified.
- the method for producing the protein having the dsRNA-degrading activity of the present invention may be a method comprising converting codons into those suitable for expression in a host or reinforcing the rare codons.
- a method in which a part or all of the amino acid sequence of the protein may be converted to an optimal codon state for protein expression, or a production method including codon modification of an expressed gene. It can be expressed using a host in a similar state.
- Optimal codon state or equivalent The host of the present invention is not particularly limited, and examples thereof include a host in which the amount of tRNA recognizing a specific codon is several times or more higher than that normally produced in cells by genetic engineering.
- Escherichia coli such as Escherichia coli supplemented with tRNAs of arginine codons (AGA, AGG), and Escherichia coli supplemented with tRNAs of isoleucine (AUA), proline (CCC), and leucine (CUA). Is mentioned.
- a method for improving the expression of the target protein in the host by performing codon modification may be used.
- the secondary structure of the mRNA may be taken into consideration.
- the method is not particularly limited as long as it is a method that improves protein expression by codon conversion, reinforcement, and the like of the expressed gene.
- any commercially available vector or expression system can be used without any particular limitation.
- a pET system manufactured by Novazidin
- a vector having a promoter that can function at a low temperature can be suitably used, and examples thereof include a pCold-based vector described in WO99 / 27117.
- a method of producing the above Dicer mutant with a vector having a promoter capable of functioning at low temperature for example, a pCold-based vector described in WO99 / 27117 is exemplified. .
- any vector in the production method of the present invention, can be suitably used as long as it can express a protein capable of retaining a function capable of producing dsRNA of a specific length.
- the protein is in the form of an inclusion body when the protein is expressed, but the function is restored by a subsequent refolding operation. Also included are vectors that can express what is possible.
- the amount of production is improved as compared with the case where the conventional full-length Dicer of human origin is expressed, and the activity of the protein is further improved.
- the acquisition rate of the holder can also be improved.
- a protein having a nucleic acid binding activity described in (2) below for example, but not limited to, RNA
- a method of expressing in the form of a fusion protein with a protein having a binding activity is also included.
- the method for promoting the degradation of dsRNA which is characterized by producing dsRNA of a specific length, according to the present invention, is performed in the presence of a protein having nucleic acid binding activity.
- the nucleic acid binding protein is not particularly limited, as long as it promotes dsRNA degradation activity.
- the above-mentioned proteins having the RNA binding activity can be suitably used.
- the protein having the RNA binding activity is not particularly limited, but examples include cold shock protein (Csp: cold shock protein).
- a cold shock protein that can function in a normal temperature range can be suitably used, and a cold shock protein derived from a thermophilic bacterium or a thermostable bacterium is preferable.
- Thermotoga maritima having the amino acid sequence IJ described in SEQ ID NO: 9 in the sequence listing (the amino acid sequence IJ encoded by the nucleotide sequence described in SEQ ID NO: 10 in the sequence listing).
- Thermotoga maritima strain MSB8 derived from Thermotoga maritima strain MSB8 was purchased from Cesamnolenk von Microorganisment sem Zelkulturen GmbH (DSM3109), and obtained from Protein Science, Vol. 8, 394.
- DSM3109 Cesamnolenk von Microorganisment selkulturen GmbH
- Recombinants can be produced by genetic engineering according to the method described on page 403 (1999).
- a vector having a promoter that can function at a low temperature can be suitably used, and examples thereof include a pCold-based vector described in WO99 / 27117.
- the dsRNA degrading activity can be promoted.
- the method of the present invention provides a protein having a dsRNA-degrading activity for producing a dsRNA of a specific length according to the above (1), for example, a functional equivalent such as a Dicer mutant, a natural Dicer or a commercially available recombinant Dicer. In either case, the activity of producing dsRNA of a specific length can be promoted.
- the protein having nucleic acid binding activity and the dsRNA degrading activity By combining the proteins, the resulting dsRNA-degrading activity can be promoted, and the resulting degradation products have a greater RNA interference effect than the degradation products obtained when the protein having no nucleic acid binding activity is not combined. It has the same activity per unit weight. Therefore, using a protein having nucleic acid binding activity is very useful in RNA interference.
- dsRNA used as a substrate is not completely degraded, whereas in the method of the present invention, substantially all of the long dsRNA serving as a substrate is cleaved. it can. Therefore, if all of the dsRNA of the substrate can be cleaved and the degradation products have the same RNA interference effect, the long dsRNA serving as the substrate will be scaled down, finned, and the undegraded dsRNA removal step will be performed. Can also be omitted.
- the protein having the nucleic acid binding activity for example, the protein having the RNA synthesis activity, may be in the form of a fusion protein with the protein having the dsRNA degrading activity.
- the method for promoting RNA synthesis of the present invention is characterized in that the method is carried out in the presence of a protein having a nucleic acid binding activity.
- the nucleic acid binding protein is not particularly limited as long as it promotes the RNA synthesis activity of the protein having the RNA synthesis activity.
- Cold shock protein (Csp: cold shock protein) ) Can be suitably used.
- the cold shock protein is not particularly limited, but a thermophilic bacterium is preferably used as the cold shock protein.
- the cold shock protein is not particularly limited, but is a CspB protein derived from Thermotoga maritima having an amino acid sequence described in SEQ ID NO: 9 in the sequence listing (base sequence described in SEQ ID NO: 10 in the sequence listing). Is exemplified.
- the coexistence of the CspB protein in the RNA synthesis system can promote, for example, the RNA synthesis activity of RNA polymerase.
- the protein can promote its synthetic activity whether the product is single-stranded or double-stranded RNA.
- the method for promoting RNA synthesis of the present invention can be used for synthesizing not only long dsRNA but also short dsRNA, for example, siRNA.
- siRNA short dsRNA
- it can be used when synthesizing preferably about 15-30 base pairs, particularly preferably about 20-25 base pairs, using T7 RNA polymerase or the like.
- the protein having nucleic acid binding activity promotes two reactions of dsRNA synthesis by a protein having RNA synthesis activity and dsRNA degradation of a protein having dsRNA degradation activity,
- it can be used for dsRNA synthesis and siRNA production systems that are important in RNA interference.
- each protein may be separate or may be in the form of a fusion protein.
- the composition of the present invention is a composition for efficiently performing a reaction to degrade into dsRNA of a specific length and / or a synthesis reaction of RNA.
- the composition includes the protein having the nucleic acid binding activity described in (2) above, and the protein having the nucleic acid binding activity is not particularly limited.
- a cold shock protein is preferable.
- a cold shock protein (Csp: cold shock protein) derived from a thermophilic bacterium or a thermostable bacterium can be suitably used, and the amino acid sequence described in SEQ ID NO: 9 in the sequence listing (encoded by the nucleotide sequence described in SEQ ID NO: 10 in the sequence listing) can be used.
- CspB protein derived from Thermotoga maritima having the following amino acid sequence:
- the composition of the present invention may contain a protein having dsRNA degradation activity and / or a protein having RNA synthesis activity.
- a protein having a dsRNA degrading activity capable of producing a dsRNA of a specific length a protein having a functional domain of Dicer is preferable.
- a protein comprising RNaseIIIa, b and a dsRNA binding domain can be suitably used.
- any of the functional equivalents such as the Dicer mutant described in (1) above, a natural Dicer, or a commercially available recombinant Dicer may be used.
- composition containing the Dicer variant a composition comprising the amino acid sequence of SEQ ID NO: 4 or 17 in the sequence listing, or a protein comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3 or 16 in the sequence listing is used. It may be a composition containing. Also, in the amino acid sequence of SEQ ID NO: 4 or 17 in the sequence listing, a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added is included. Alternatively, the composition may be.
- a control sequence derived from an expression vector for example, an expression or translation enhancing sequence (for example, Perfect DB sequence, etc.), a tag sequence for purifying an expressed protein (for example, His tag sequence, etc.) )
- a protein to which an amino acid sequence such as a sequence for removing an additional sequence at the N-terminal side of the expressed protein eg, Factor Xa sequence
- the protein include, but are not particularly limited to, a protein having an amino acid sequence represented by SEQ ID NO: 12 or 18 in the sequence listing and having a dsRNA degrading activity.
- the composition of the present invention may contain a buffer for stabilizing the Dicer mutant described in (1) above.
- RNA polymerase for example, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase and the like can be suitably used.
- a protein having the nucleic acid binding activity described in the above (2) for example, a protein having an RNA binding activity and having a dsRNA degrading activity capable of producing dsRNA of a specific length It may contain a fusion protein with a protein and / or a fusion protein of a protein having an activity of binding to a nucleic acid and a protein having an activity of synthesizing an RNA.
- composition of the present invention can easily perform efficient degradation into dsRNA of a specific length and / or synthesis reaction of single-stranded or double-stranded RNA.
- composition of the present invention includes not only long dsRNA but also short dsRNA, for example, a composition that can be used for the synthesis of siRNA.
- the composition is effective in synthesizing dsRNA of about 10-100 base pairs, preferably about 15-30 base pairs, particularly preferably about 20-25 base pairs.
- the kit used in the method of the present invention is a kit for efficiently performing a reaction for decomposing into dsRNA of a specific length and a reaction for synthesizing Z or RNA.
- the kit includes the protein having the nucleic acid binding activity described in (2) above, and the protein having the nucleic acid binding activity is not particularly limited.
- a cold shock protein is preferable.
- Cold Shock Pro derived from thermostable bacteria Tin (Csp: cold shock protein) can be suitably used, and Cs pB protein derived from Thermotoga maritima having the amino acid sequence of SEQ ID NO: 9 in the sequence listing can be suitably used.
- the kit of the present invention may include a protein having an activity of producing dsRNA of a specific length, a protein having a dsRNA degrading activity, and a protein having a Z or RNA synthesis activity.
- a protein having the dsRNA degrading activity and / or the protein having the RNA synthesizing activity those mentioned in the above (3) can be suitably used.
- the kit of the present invention may contain a buffer for stabilizing the Dicer mutant described in (1) above.
- a protein having the nucleic acid binding activity described in (2) above for example, a protein having an RNA binding activity and a dsRNA degrading activity capable of producing dsRNA of a specific length are provided. It may contain a fusion protein with a protein and a fusion protein of a protein having Z or nucleic acid binding activity and a protein having RNA synthesis activity.
- the kit of the present invention may contain components other than those described above, for example, a reagent for purifying dsRNA of a specific length generated as a result of the reaction, a reagent for introducing the same into a biological sample, and the like. good. Although not particularly limited, it may contain, for example, a reagent for purifying about 21 base pairs of siRNA, a reagent for introducing the same into a living sample, and the like.
- kits of the present invention By using the kit of the present invention, efficient degradation into dsRNA of a specific length and / or synthesis of RNA can be easily performed.
- kits of the present invention includes a kit that can be used for synthesizing not only long dsRNA but also short dsRNA, for example, siRNA.
- the kit is effective in synthesizing dsRNA of about 10 to 100 base pairs, preferably about 15 to 30 base pairs, particularly preferably about 20 25 base pairs.
- an expression vector was constructed as follows.
- synthetic primers 1 and 2 having the nucleotide sequences of SEQ ID NOs: 5 and 6 in the Sequence Listing were synthesized using a DNA synthesizer based on the nucleotide sequences disclosed in Gene Bank Registration No. AB028449, and purified by a conventional method.
- the synthetic primer 1 has a nucleotide sequence corresponding to amino acid numbers 1271-1277 of the amino acid sequence of human-derived Dicer (SEQ ID NO: 1) at base numbers 16-36, It is a synthetic DNA with.
- synthetic primer 2 had a base sequence corresponding to amino acid numbers 1919 to 1924 of the amino acid sequence of human-derived Dicer (SEQ ID NO: 1) at base numbers 911 to 1414, — At 36.
- PCR was performed using the above synthetic primers.
- the reaction conditions for PCR are shown below.
- type II DNA human cDNA library, Human Pancreas, manufactured by Takara Bio Inc. 2 / i1, 5 / i 1 of 10X LA PCR buffer (manufactured by Takara Bio Inc.), 5 ⁇ l of dNTP mixed solution (Takara Bio Inc.) Bio Primer), l Opmol Synthetic Primer 1, l Opmol Synthetic Primer 2, 0.5 U Takara LA Taq (Takara Bio) and sterilized water to make a total volume of 50 ⁇ l.
- the reaction solution was set on a TaKaRa PCR Thermal Cycler SP (manufactured by Takara Bio Inc.), and the temperature was 94 ° C for 1 minute, 55 ° C for 1 minute, and 72. A 30-cycle reaction was performed with one cycle of C 3 minutes.
- the pCold08NC2 vector was prepared according to the method described in Example 16 of WO99Z27117 pamphlet.
- the pCold08 vector was cleaved with the same restriction enzymes used when preparing the Kpnl-Hinddlll digested DNA fragment, and the one obtained by dephosphorizing the end was prepared, mixed with the KpnI_HindIII digested DNA fragment, Ligation was performed using a DNA ligation kit (manufactured by Takara Bio Inc.). Then, Escherichia coli JM109 was transformed using 20 ⁇ l of the ligation reaction mixture, and the transformant was grown on LB medium (containing 50 ⁇ g / ml ampicillin) containing 1.5% (wZv) agar. I let it.
- the plasmid into which the desired DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold08hDi-R.
- the plasmid is named and designated as pCold08hDi-R, and from August 11, 2003 (Hara Deposit Date), the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba East 1-chome, Ibaraki, Japan) Deposited as FERM BP-10074 at 1 No. 1 Central No. 6 (Zip Code 305-8566).
- This pCold 08 hDi-R is a plasmid containing a nucleotide sequence encoding the amino acid sequence of amino acids 1271-2924 of the Dicer amino acid sequence of human origin (SEQ ID NO: 1).
- the protein expressed from the plasmid has Perfect DB system U, His tag sequence, and Factor Xa system IJ.
- the amino acid sequence of the protein is shown in SEQ ID NO: 12 in the sequence listing, and the base sequence is shown in SEQ ID NO: 13 in the sequence listing.
- Escherichia coli BL21 was transformed with pCold08 hDi-R prepared in (1) above, and the transformant was transformed into an LB medium containing 1.5% (w / v) agar (containing 50 ⁇ g / ml ampicillin). Mu).
- the grown colonies were inoculated into 2.5 ml of an LB liquid medium (containing 50 xg / ml of ampicillin) and cultured at 37 ° C. A portion of this was inoculated into 100 ml of the same LB medium and cultured at 37 ° C until logarithmic growth. Incubator kept at 15 ° C after the culture After shaking for 10 minutes in one tube, IPTG was added to a final concentration of 1.
- buffer A 20 mM Tris-HCl buffer (pH 7.5), 100 mM sodium chloride, ImM dithiolate, 0.1% Triton X-100 was added and mixed, and then centrifuged at 1,500 rpm for several minutes. The supernatant was discarded, and about 1 ml of the resin was recovered. About 5 ml of the supernatant prepared from the cell lysate was added to the mixture, and the mixture was gently mixed on a rotary shaker at 4 ° C for about 1 hour. Thereafter, the resin to which the target protein was adsorbed was packed in a ⁇ 15 mm column, and washed twice with 5 ml of buffer A.
- buffer B 20 mM Tris-HCl buffer ( ⁇ 7.5), 100 mM sodium chloride, ImM dithylate latex, 0.1% Triton X_100, 40 mM imidazole, and then washed with 5 ml.
- BufferC 20mM Tris-HCl buffer (pH 7.5), 800mM sodium chloride, ImM dithiothreitol, 0.1% Triton X_100, 40mM imidazole
- Unnecessary proteins other than the purpose were removed.
- bufferD 20 mM Tris-hydrochloric acid buffer (pH 7.5), lOOmM sodium chloride, ImM dithiothrein monol, 0.1% Triton X-100, lOOmM imidazole].
- buffer E 50 mM Tris-monohydrochloride buffer (pH 8.0), 100 mM salt solution]; pam, 0.5 mM EDTA, 0.1% toluene X_100, lm M dithiothre And then concentrated to about 10-fold using Centricon (manufactured by Amicon). For a portion of this purified concentrated sample 10.
- hDiR human-derived Dicer RNaselll domain protein
- Dicer activity of the protein samples I-IV prepared in Example 11- (2) above was measured. The activity was measured as follows.
- dsRNA as a substrate used for activity measurement was synthesized using TurboScript T7 Transcription kit (manufactured by GTS) according to the protocol attached thereto.
- dsRNA having a length of about 700 bp was prepared by performing an RNA synthesis reaction using T7 RNA polymerase using the obtained double-stranded DNA as type II.
- a reaction solution was prepared by adding 2 ⁇ l of the buffer solution used for dialysis, which was 5 times concentrated, and adding nuclease free water to make a total volume of 10 ⁇ l.
- Dicer manufactured by GTS
- 2 ⁇ l of Dicer enzyme solution 1 ⁇ l of dsRNA lzgl OmM ATP solution as a substrate, 1 ⁇ m at night, and 0.5 ⁇ m of 50 mM salted magnesium solution at night 1.
- the attached reaction buffer (4 ⁇ l) was added with nuclease-free water to make a total volume of 10 ⁇ l.
- the activity of the reaction solution prepared in the above (1) was measured, and the influence on the RNaselll domain protein (hDiR) was examined. Furthermore, in order to examine the stability in each buffer solution, a sample immediately after purification and a sample stored at 4 ° C. and 120 ° C. for 5 days were used. As a result, especially in protein sample III and protein sample IV, degradation products of about 21 nucleotides, the same size as the commercially available Dicer, were confirmed at both 4 ° C storage and -20 ° C storage. It was confirmed that the activity was stably maintained.
- Thermotoga maritima having the amino acid sequence of SEQ ID NO: 9 in the Sequence Listing tima) -derived CspB protein was used as a model protein.
- the protein was prepared by the method described in Protein Science, Vol. 8, pages 394-403 (1999).
- the dsRNA degradation activity in the form to which CspB was added was measured as follows.
- Example 2_ 1 ⁇ l of dsRNA lzgl OmM ATP solution used in 1), 1 ⁇ m of 50 mM salted magnesium solution, 5 ⁇ reaction buffer [250 mM Tris-hydrochloric acid (pH 8.5), 500 mM salted sodium, 0.5 mM % TritonX-100, 5 mM DTT] 2 ⁇ l, and nuclease-free water was added to make a volume of 10 ⁇ l, and this was used as a reaction solution.
- Dicer In the case of commercially available Dicer, 1 ⁇ g of dsRNA serving as a substrate was added to the composition described in the attached document, and nuclease-free water was added thereto to make the volume 10 ⁇ l.
- dsRNA dsRNA serving as a substrate
- nuclease-free water was added thereto to make the volume 10 ⁇ l.
- commercially available Dicer those manufactured by GTS, Stratagene and Invitrogen were used.
- the added CspB protein was added at a final concentration of 4.6 ng / ⁇ 1 , 9.2 ng // i1, 18.4 ng // il, and 92 ⁇ / ⁇ 1.
- 1 ⁇ 1 was added to a 10 mM potassium phosphate buffer (pH 7.5) as a CspB shape buffer.
- RNA interference based on the view that it is also important to promote the activity of the RNA synthesis system to synthesize dsRNA, the effect of Thermotoga maritima-derived CspB on RNA synthesis was examined.
- the T7 RNA polymerase system was selected as a model system.
- the effect on the RNA synthesis system was performed as follows. That is, the amount of transcription by T7 RNA polymerase in the form in which CspB was added was used as a standard.
- a plasmid prepared by introducing the rsGFP gene having the nucleotide sequence of SEQ ID NO: 11 into pET16b (manufactured by Novadin) having a T7 promoter prepared by a conventional method as 1 ⁇ g type 10 DNA for 10X T7 RNA polymerase Buffer solution (Takara Bio Inc.) 2 ⁇ 1, 50 ⁇ DTT 2 ⁇ 1, RNase inhibitor (Takara Bio Inc.) 0.4 ⁇ 1, 25 mM NTP 2 ⁇ 1, T7 RNA polymerase (Takara Bio Inc.) 1 ⁇ 1.
- the mRNA synthesized by T7 RNA polymerase was quantified by image analysis of the gel using Total Lab ver. 1.11 (Nonlinear Dynamics). As a result, it was confirmed that the transcript amount was improved at any concentration. In particular, it was confirmed that when the added amount of CspB was 460 ng Zxl or more, the amount of the transcript was about twice or more than that when no addition was performed, and about 3 times when the addition of 920 ng / z 1 was further added. .
- Example 2_ (1) the ⁇ type for dsRNA synthesis prepared in Example 2_ (1) was used.
- a commercially available TurboScript T7 Transcription kit manufactured by Gene Therapy Systems
- the final concentration of CspB protein added at this time was 90 ng / ⁇ 1, 180 ng / ⁇ 1, 460 ng / ⁇ 1, and 920 ng Zxl.
- a 10 mM potassium phosphate buffer solution (pH 7.5) was added in a single addition.
- dsRNA synthesis type 1 prepared in Example 2_ (1), 1 ⁇ l of 10 XT7 RNA polymerase buffer (manufactured by Takara Bio Inc.), 50 mM DTT 1 / i 1, RNase inhibitor (Takara Bio Inc.) 0.2 ⁇ 1, 25 ⁇ ⁇ 1 / i1, ⁇ 7 RNA polymerase (manufactured by Takara Bio Inc.) 0.5 xl, CspB solution 1 / i1, and add nuclease free water to make the volume 10 ⁇
- the reaction mixture was used as a reaction solution and reacted at 37 ° C. for 4 hours.
- the concentration of C spB protein / protein added at this time should be 90 ng / ⁇ 1, 180 ng / ⁇ 1, 460 ng / ⁇ 1, and 920 ng // i1 in the final concentration.
- 1 ⁇ m potassium phosphate buffer ( ⁇ 7.5) which is a CspB shape buffer, was added.
- DNas el manufactured by Takara Bio Inc.
- 1 ⁇ l of a 40-fold diluted solution of this reaction solution was subjected to 1% agarose gel electrophoresis containing ethidium bromide.
- the gel was also quantified for dsRNA. As a result, it was confirmed that the transcript amount was improved at any concentration. In particular, when the added amount of CspB was 920 ng / zl or more, it was confirmed that the amount of the transcript was about three times or more than that in the case of the untreated kafun.
- an expression vector was constructed as follows.
- synthetic primers 5 and 6 having the nucleotide sequences of SEQ ID NOs: 14 and 15 in the sequence listing were synthesized using a DNA synthesizer, and purified by a conventional method.
- the above-mentioned synthetic primer 5 has a nucleotide sequence corresponding to amino acid number 679-685 of the amino acid sequence of human-derived Dicer (SEQ ID NO: 1) at nucleotide number 914, and a nucleotide sequence at nucleotide number 916, which is a recognition sequence of restriction enzyme Kpnl. — Synthetic DNA of 36.
- synthetic primer 6 has a base sequence corresponding to amino acid numbers 1919 to 1924 of amino acid sequence 1J (SEQ ID NO: 1) of human-derived Dicer, and a base sequence corresponding to the recognition sequence of restriction enzyme Hindlll at base numbers 911 to 1414. 18-35.
- PCR was performed using the above synthetic primers.
- the reaction conditions for PCR are shown below.
- type II DNA human cDNA library, Human Pancreas, manufactured by Takara Bio Inc. 2 / i1, 5 / i 1 of 10X LA PCR buffer (manufactured by Takara Bio Inc.), 5 ⁇ l of dNTP mixed solution (Takara Bio Inc.) Bio Primer), l Opmol synthetic primer 5, l Opmol synthetic primer 6, 0.5 U of Takara LA Taq (manufactured by Takara Bio Inc.), and sterilized water were added to make a total volume of 50 / i 1.
- the reaction solution was set on a TaKaRa PCR Thermal Cycler SP (manufactured by Takara Bio Inc.), and 30 cycles of reaction were performed at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 3 minutes.
- Example 1_ (1) the pCold08NC2 vector prepared in Example 1_ (1) was digested with Kpnl-Hindll as described above.
- a DNA fragment was digested with the same restriction enzymes used when the DNA fragment was prepared, dephosphorylated, and mixed with the above Kpnl-Hindlll digested DNA fragment, and a DNA ligation kit (Takara Bio Inc.) Were connected.
- Escherichia coli JM109 was transformed using the ligation reaction mixture 20/1, and the transformant was grown on LB medium (containing 50 ⁇ g Zml of ampicillin) containing 1.5% (w / v) agar.
- LB medium containing 50 ⁇ g Zml of ampicillin
- the plasmid into which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold08hDi_ASI.
- the plasmid is named and designated as pCold08hDi-ASI, and from September 26, 2003 (Hara Deposit Date) Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba East 1-chome, Ibaraki, Japan) Address No. 1 Central No. 6 (Zip code 305-8566) is deposited as FERM BP-10076.
- This pCold08 hDi-ASI is the base sequence described in SEQ ID NO: 16 and SEQ ID NO: 17 in the base sequence IK sequence listing which encodes the amino acid sequence of amino acids 679 to 1924 in the Dicer amino acid sequence of human origin (SEQ ID NO: 1).
- the protein expressed by the plasmid has the Perfect DB system, His tag sequence, and Factor Xa sequence.
- the amino acid sequence of the protein is shown in SEQ ID NO: 18 in the sequence listing, and the nucleotide sequence is shown in SEQ ID NO: 19 in the sequence listing.
- Escherichia coli BL21—CodonPlus-RI L strain (Stratagene) was transformed, and the transformant was concentrated at 1.5% (w / v). It was grown on LB medium containing ampicillin (containing 50 ⁇ g / ml ampicillin). The grown colonies were inoculated into 2.5 ml of LB liquid medium (containing 50 ⁇ g / ml of ampicillin) and cultured at 37 ° C. A portion of this was inoculated into 100 ml of the same LB medium and cultured at 37 ° C until logarithmic growth.
- the cells were shaken in an incubator kept at 15 ° C for 10 minutes, IPTG was added to a final concentration of 1.0 mM, and the cells were cultured at 15 ° C for 24 hours to induce expression.
- the cells were then collected by centrifugation, and 5 ml of the cell disruption solution [50 mM Tris monochloride buffer ( ⁇ 8.5), lOOmM sodium chloride, ImM magnesium chloride, 0.1% Triton X_100, ImM dithiothreitol , ImM phenylmethylsulfonyl fluoride].
- the cells are disrupted by sonication and centrifuged (11, OOOrpm (20 minutes) to separate the supernatant into an extract and a precipitate.
- buffer A 20 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, ImM dithiothreitol, ImM magnesium chloride, 0.1% Triton X were added to Ni_NTA agarose (manufactured by QIAGEN) for 1 ml of resin volume. Then, the mixture was centrifuged at 1,500 rpm for several minutes, the supernatant was discarded, and about 1 ml of the resin was recovered. About 5 ml of the supernatant prepared from the cell lysate was added and gently mixed on a rotary shaker at 4 ° C for about 1 hour.
- the resin to which the target protein was adsorbed was filled in a ⁇ 15 mm column, and washed twice with 5 ml of buffer A.
- wash the resin with 5 ml of buffer B [20 mM Tris monochloride buffer ( ⁇ 8.5), 100 mM sodium chloride, ImM magnesium chloride, ImM dithiothreitol, 0.1% Triton X_100, 40 mM imidazole], and then wash 5 ml of the resin.
- BufferC 20 mM Tris-hydrochloride buffer ( ⁇ 8.5), 800 mM sodium chloride, ImM magnesium chloride, ImM dithiothreitol, 0.1% Triton X_100, 40 mM imidazole], followed by 5 ml buffer B After washing, unnecessary proteins other than those intended were removed.
- bufferD 20 mM Tris-hydrochloric acid buffer (pH 8.5), lOOmM sodium chloride, ImM magnesium chloride, ImM dithiothreitol, 0.1% Triton X-100, lOOmM imidazole
- concentration was performed to about 10-fold using Centricon (manufactured by Amicon).
- hDi_ASI human-derived Dicer PAZ + RNaseIII domain protein
- a degradation product of about 21 nucleotides was confirmed in the gel stained with ethidium bromide after electrophoresis, and was found in the RNaselll domain protein (hDiR) and the protein containing the PAZ region (hDi-ASI). Also, dsRNA degradation activity was confirmed.
- Example 4-(2) For the hDi-ASI prepared in Example 4-(2) above, the effect of a protein having nucleic acid binding activity at room temperature was examined.
- the CspB protein derived from Thermotoga maritima prepared in Example 3 was used as the protein having the nucleic acid binding activity.
- the effect on dsRNA degradation was measured as follows.
- the concentration of CspB protein was adjusted to a final concentration of 9.2 ng / zl, 18.4 ng / ⁇ l, and 92 ng / ⁇ l, and as a control in the case of no addition, 10 mM phosphorus, a CspB shape buffer, was used. Potassium acid buffer (pH 7.5) was added: 1. After preparing the above reaction solution and reacting at 37 ° C. for 17 hours, 5 / l was subjected to 15% polyacrylamide gel electrophoresis and stained with ethidium bromide to confirm the cleavage product. Furthermore, the genole was analyzed by image analysis using Total Lab ver. 1.11 (Nonlinear Dynamics) to quantify dsRNA degradation products of about 21 nucleotides.
- CspB promotes its dsRNA degradation activity even in the case of natural or mutant human-derived Dicer containing an RNaselll domain.
- RNA interference of siRNA prepared using the human-derived hDiR of the present invention was examined.
- a commercially available Dicer GTS
- Preparation of the dsRNA degradation product was basically performed by the method described in Example 2- (1) above. That is, using 10 units of the hDiR described in Example 1_ (2) and a commercially available Dicer, 10 ⁇ g of dsRNA was cut at 37 ° C. for 18 hours. These cleavage products were purified using RNA Purification Columns 1 and 2 (manufactured by Gene Therapy Systems) and used for the following evaluation of RNA interference.
- TransIT 293 Transfection Reagent (manufactured by TAKARA BIO INC.) was added to 50 / il serum-free medium and stirred vigorously. After leaving at room temperature for 5 minutes, 0.3 / g of pQBI25 (manufactured by Wako Pure Chemical Industries, Ltd.) was added, mixed gently, and left at room temperature for 5 minutes. 4 ⁇ l of TransIT-TKO reagent was added thereto, mixed gently, and left at room temperature for 5 minutes. Then, 500 ng of the above siRNA was added thereto, mixed gently, and allowed to stand at room temperature for 5 minutes to prepare a DNA / siRNA solution.
- the DNA / siRNA solution was added dropwise to the D-MEM medium containing 10% FBS in the wells at a concentration of 250 ⁇ l, and mixed gently so that the solution in the wells became uniform.
- a sample containing only pQBI25 manufactured by Wako Pure Chemical Industries, Ltd.
- a sample containing only sterile water were also performed. Then CO
- RNA interference occurs as the average fluorescence value decreases as compared with the control (vector only). Therefore, it was confirmed that the siRNA obtained by hDiR exhibited an RNA interference effect similarly to that of the commercially available Dicer, and showed a stronger level of RNA interference than that of the commercially available Dicer.
- the hDiR of the present invention is useful for preparing siRNA for RNA interference.
- siRNA prepared using the hDiR of the present invention were examined by changing the amount of siRNA added.
- a commercially available Dicer manufactured by Gene Therapy Systems
- the cleavage of dsRNA was basically performed by the method described in Example 2_ (1) above. That is, 10 ⁇ g of dsRNA was cut at 37 ° C. for 18 hours using 10 ⁇ l of the hDiR described in Example 11 (2) and a commercially available Dicer. In this case, CspB used in Example 3- (2) was added to the final concentration of 9.2 ng / xl, and reacted.
- RNA Purification Columns 1 and 2 Gene Therapy Systems
- a separate tube prepared by adding 3 ⁇ l of Ribojuice Transfection Reagent (manufactured by Takara Bio Inc.) to 47 ⁇ l of a serum-free medium was prepared and stirred vigorously. After leaving at room temperature for 5 minutes, 166.7 ng, 55.6 ng and 18.5 ng of the above siRNA were added, mixed gently, and allowed to stand at room temperature for 5 minutes.
- the two solutions prepared in this way were used in wells. /.
- the D-MEM medium containing FBS was added dropwise to the medium to which the concentration had been adjusted to 250 ⁇ ⁇ , and the mixture was gently mixed so that the solution in the well became uniform.
- a sample to which only a vector (DNA) was added and a sample to which only sterilized water was added were also used. Then in the CO incubator 24
- Control door (vector only) 1 0 0
- RNA interference occurs as the value of the average fluorescence intensity is smaller as compared with the control (vector only). Therefore, it was confirmed that the siRNA obtained by hDiR exhibited an RNA interference effect in the same manner as the commercial Dicer.
- the hDiR of the present invention was useful for preparing siRNA for RNA interference.
- RNA interference was evaluated by extracting total RNA from the above cell sample and subjecting it to real-time RT-PCR to quantify rsGFP mRNA. [0091] That is, after cells were cultured at 37 ° C for 24 hours in a CO incubator after siRNA introduction,
- reaction solution was set in TaKaRa PCR Thermal Cycler SP (manufactured by Takara Bio Inc.), and reacted at 42 ° C for 10 minutes and at 95 ° C for 2 minutes.
- 20 ⁇ of a reaction diluent IX M_MLV buffer, 0.5 mM dNTP mixture
- 10XR-PCR buffer 2.5 ⁇ 1, 250mM Mg 2+ 0.3 ⁇ ⁇ lOmM dNTP 0.75 ⁇ 1, TaKaRa Ex Taq R—PCR (Takara Bio Inc.) 1.
- rsGFP_F SEQ ID NO: 20
- rsGFP-R SEQ ID NO: 21
- Neo Neo_F: SEQ ID NO: 22, Neo-R: SEQ ID NO: 23
- the reaction solution was set in a Smart Cycler II Unit (manufactured by TAKARA BIO INC.), Heat denatured at 95 ° C for 10 seconds, and then 95 cycles at 5 seconds at 95 ° C and 20 cycles at 60 ° C for 45 cycles was carried out.
- TAKARA BIO INC. Smart Cycler II Unit
- the mRNA levels of human-derived i3_actin and GAPDH, and rsGFP and Neo derived from the introduced plasmid were quantified. The results are shown in Table 3.
- RNA interference occurs as the amount of rsGFP mRNA is smaller than that of the control (vector only). Therefore, it was confirmed that hDiR exhibited an RNA interference effect similarly to the retail Dicer.
- the hDiR of the present invention was useful for preparing siRNA for RNA interference.
- dsRNA as a substrate for dsRNA degradation activity was prepared from the luciferase gene and evaluated.
- dsRNA was synthesized using TurboScript T7 Transcription kit (manufactured by GTS) according to the protocol attached thereto.
- the plasmid pGL3_Basic vector (Promega) is used as a type III, and the T7 promoter described in SEQ ID NO: 24 in the sequence listing is used.
- PCR amplified fragment length: about 500 base pairs
- dsRNA with a length of about 500 bp was prepared by using the obtained double-stranded DNA as ⁇ type and performing RNA synthesis reaction with T7 RNA polymerase.
- RNA interference effect of siRNA prepared using the hDiR and hDi-ASI of the present invention was examined.
- a commercially available Dicer (Gene Therapy Systems) was used as a control.
- the cleavage of dsRNA was basically performed by the method described in Example 2- (1) above. That is, using the hDiR described in Example 11- (2), the hDi-ASI described in Example 4-1- (2), and a commercially available Dicer ⁇ , the above-mentioned dsRNA10 ⁇ g was converted to 37 ° C. C, cut in 18 hours. In this case, the CspB used in Example 3- (2) was added with calorie so as to have a final concentration of 9.2 ngZ ⁇ l, and reacted.
- RNA Purification Columns 1 and 2 Gene Therapy Systems
- a separate tube prepared by adding 47 ⁇ l of serum-free medium to 3 ⁇ l of Ribojuice Transfection Reagent (Takara Bio Inc.) was prepared and stirred vigorously. The mixture was allowed to stand at room temperature for 5 minutes, and the above-mentioned siRNA was transcribed without calorie from 166.7 ng, 55.6 ng, and 18.5 ng, and allowed to stand at room temperature for 5 minutes.
- the two solutions prepared in this way were added to the wells 10. /.
- the D-MEM medium containing FBS was added dropwise to the medium to which the concentration had been adjusted to 250 ⁇ ⁇ , and the mixture was gently mixed so that the solution in the well became uniform.
- a sample to which only the vector (DNA) was added and a sample to which only sterilized water was added were also performed at the same time. Then in the CO incubator 24
- siRNA sample GL3 expression level relative value control (no addition) ⁇
- hDiR and hDi-ASI of the present invention were useful for preparing siRNA for RNA interference.
- RNA interference was examined for the siRNA prepared by adding CspB and the siRNA prepared without the addition.
- the actual operation was learned from the method using luciferase described in Example 7.
- Commercially available Dicer (Gene Therapy Systems) was used.
- Cleavage of dsRNA was basically performed by the method described in Example 2- (1) above. That is, using the hDiR prepared by the method described in Example 1- (2), the hDi-ASI prepared by the method described in Example 4_ (2), and Cut at 18 ° C for 18 hours. Note that at this time, was reacted with the case where the CspB used was added to a final concentration 9.2 ⁇ ⁇ / ⁇ 1 in Example 3- (2) added Shinare, if the.
- RNA Purification Columns 1 and 2 Gene Therapy Systems
- the CspB of the present invention was not affected qualitatively.
- Example 4_ The PAZ domain + RNase III domain protein (hDi-ASI) purified in (2) and CspB added to a final concentration of 92 ng / ⁇ l were added to storage buffer I (50 mM Tris-HCl buffer ( pH 8.5), stored in 250 mM sodium chloride, ImM magnesium chloride, 0.1 mM DTT, 0.1% TritonX-100, 50% glycerol), and its dsRNA degradation at regular intervals as follows: Activity was measured.
- Example 2 1 ⁇ g of dsRNA prepared in (1), 1 ⁇ l of protein sample prepared in 4_ (2) above, 5X reaction buffer (100 mM Tris-HCl buffer (pH 8.5), 750 mM chloride Sodium, 12.5 mM magnesium chloride) 2 ⁇ l, and nuclease free water was added to make a total volume of 10 ⁇ l, which was used as a reaction solution. After reacting at 37 ° C for 18 hours, 5 was subjected to 15% polyacrylamide gel electrophoresis and staining with ethidium bromide to confirm the cleavage product. As a result, degradation products of about 21 base pairs were confirmed in the stored samples for 6 months or longer, and dsRNA degradation activity was confirmed. On the other hand, under the same buffer conditions, the activity of RNaselll domain protein (hDiR) plus CspB was maintained for 3 months or more.
- hDiR RNaselll domain protein
- TmCspB Thermotoga maritima
- synthetic primers E and F having the nucleotide sequences of SEQ ID NOs: 26 and 27 in the sequence listing were synthesized using a DNA synthesizer, and purified by a conventional method.
- the synthetic primer E has a nucleotide sequence corresponding to the amino acid sequence 117 of the amino acid sequence 1J (SEQ ID NO: 26) of the amino acid sequence 1J (SEQ ID NO: 26) of the recognition sequence of the restriction enzyme Ndel, 34 is a synthetic DNA of 34.
- Synthetic primer F has a base sequence corresponding to amino acids 61-66 of the amino acid sequence of TmCspB (SEQ ID NO: 26) at base Nos. DNA. [0105] PCR was performed using the above synthetic primers.
- the reaction conditions for PCR are shown below. That is, type 3 DNA (50 ng) of Example 3_ (1), 5 / il lOX Ex Taq buffer (manufactured by Takara Bio Inc.), 5 / il dNTP mixed solution (manufactured by Takara Bio Inc.), synthetic lOpmol Ima-E, lOpmol of synthetic primer F, 0.5 U of Takara Ex Taq (manufactured by Takara Bio Inc.) were added, and sterile water was added to make a total volume of 50 ⁇ .
- the reaction solution was set on a TaKaRa PCR Thermal Cycler SP (manufactured by Takara Bio Inc.), and set at 94 ° C. for 1 minute, 55. The reaction was performed for 30 cycles, each cycle consisting of C1 minute and 72 ° C1 minute.
- reaction solution 5 was subjected to 3.0% agarose gel electrophoresis to confirm a target DNA fragment of about 22 Obp.
- the remaining PCR reaction solution was subjected to electrophoresis, the fragments were collected, purified, and ethanol precipitated.
- the recovered DNA after ethanol precipitation was suspended in 5 ⁇ l of sterile water, double digested with the restriction enzyme Ndel (Takara Bio Inc.) and the restriction enzyme BamHI (Takara Bio Inc.), and subjected to 3.0% agarose electrophoresis.
- the NdeI_BamHI digest was extracted and purified to obtain an Nde-BamHI digested DNA fragment.
- pCold04NC2 vector was prepared according to the method described in Example 16 of WO 99/27117 (hereinafter, this pCold04NC2 vector is referred to as pColdl4 vector).
- the above pColdl4 vector was cleaved with the same restriction enzymes used when preparing the above DNA fragment, and the one obtained by dephosphorizing the end was prepared, and mixed with the above Nde to BamHI digested DNA fragment. And ligated using a DNA ligation kit (manufactured by Takara Bio Inc.). Then, Escherichia coli JM109 was transformed using 20 ⁇ l of the ligation reaction mixture, and the transformant was transformed into an LB medium containing agar at a concentration of 1.5% (w / v) (containing 50 ⁇ g / ml ampicillin). Grown on top.
- the plasmid into which the target DNA fragment was inserted was confirmed by sequencing.
- Escherichia coli BL21 was transformed using pColdl4-TmCspB prepared in Example 10- (1) above, and the transformant was transformed into an LB medium containing 1.5% (wZv) agar in LB medium (ampicillin 50 ⁇ m). g / ml). The grown colonies are transferred to a 30 ml LB liquid medium (bact 0.3 g of o-tryptone, 0.15 g of bacto-yeast extract, 0.15 g of NaCl, and 1.5 mg of ampicillin), and cultured at 37 ° C for 1 hour.
- the cells were collected by centrifugation to obtain 11 lg of wet cells.
- the wet bacterial cells were resuspended in 44 ml of buffer A [20 mM Tris-HCl buffer (pH 8.0)].
- the cells were disrupted by sonication and separated into a supernatant extract and a precipitate by centrifugation (10,000 ⁇ g, 20 minutes).
- the supernatant extract was further separated into a supernatant extract and a precipitate by ultracentrifugation (70,000 ⁇ g for 20 minutes).
- the supernatant and the precipitate were separated by centrifugation (3,000 X g for 20 minutes), and the supernatant was further filtered through a glass filter. 45 ml of the filtrate was dialyzed against 3 liters of buffer A [20 mM Tris-HCl buffer (pH 8.0)].
- the fraction containing the target protein with a molecular weight of about 7,500 confirmed by Tricine-SDS polyacrylamide electrophoresis was collected and dialyzed against 3 liters of bufferA [20 mM Tris-HCl buffer ( ⁇ 80)].
- bufferA 20 mM Tris-HCl buffer ( ⁇ 80)].
- 65 ml of the dialyzed solution was concentrated up to about 144 times using UF3,000 cut Centriprep YM-3 (manufactured by Millipore) to obtain about 450 protein samples.
- An equal amount of glycerol was added to this protein sample to obtain 850 ⁇ l of a 50% glycerol solution (WZW).
- WZW 50% glycerol solution
- Example 10 1 ⁇ l of the protein sample (hDi-R enzyme solution) or 1 ⁇ l of the protein sample (hDi-ASI enzyme solution) prepared in Example 11 (2) and Example 4 (2), and Example 10— ( 1 / i 1 of the CspB solution (92 ng / ⁇ 1) prepared in 2), 1 g of dsRNA as a substrate prepared in Example 2- (1), 5X reaction buffer (100 mM Tris-HCl buffer ( ⁇ 8 ⁇ 5), 750 mM sodium chloride, 12.5 mM magnesium chloride) 2 / i1, and nuclease-free water was added to make a total volume of 10 ⁇ 10, and the reaction solution was used.
- the final concentration of CspB protein is 9.2 ng // il.
- a commercially available Dicer was also used as a control in this case.
- 2 ⁇ l of enzyme solution, 1 n1 of CspB solution, dsRNA l / ig as a substrate, 10 mM ATP solution 1 / i 1, 50 mM salted magnesium solution 0.5 ⁇ 1, the attached reaction buffer 41, and nuclease-free water were added to make a volume of 10 ⁇ 1 to obtain a reaction solution.
- a negative control was prepared by adding 1 ⁇ l of nuclease free water instead of TmCspB.
- Example 10- (2) After preparing the above reaction solution and reacting at 37 ° C for 18 hours, 5 was subjected to 15% polyacrylamide electrophoresis and stained with ethidium bromide to confirm the cleavage product. The resulting DNA was analyzed by image analysis using Total Lab ver. 1.11 (Nonlinear Dynamics) to quantitate dsRNA degradation products of about 21 nucleotides. As a result, it was confirmed that TmCspB expressed and purified in Example 10- (2) had an improved degradation amount of dsRNA, similarly to TmCspB expressed and purified in Example 3- (1).
- E. coli BL21 was transformed using pCold08 hDi-ASI prepared in Example 4 (1) above, and the transformant was transformed into an LB medium (ampicillin 50) containing agar at a concentration of 1.5% (wZv). ⁇ g / ml).
- the grown colonies were inoculated into 200 ml of TB liquid medium (bacto-tryptone 2.4 g, bacto-yeast extract 4.8 g, glycerol 0.8 mi, 1 / mM KHP ⁇ , 72 mM KHP ⁇ , ampicillin lOmg). Fungus and 37.
- a portion of 12 g of the wet cells was treated with 48 ml of a cell disruption solution [50 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, 1 mM magnesium chloride, protease inhibitor (Complete, EDTA-free, Boehringer Mannheim)] ].
- the cells were disrupted by sonication and separated into a supernatant extract and a precipitate by centrifugation (12, OOOrpm for 30 minutes).
- the resin to which the target protein is adsorbed is packed into a ⁇ 20 mm column, and 40 ml of a cell disruption solution [50 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, ImM salt Magnesium chloride and protease inhibitor (Complete, EDTA-free, Boehringer Mannheim)].
- a cell disruption solution 50 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, ImM salt Magnesium chloride and protease inhibitor (Complete, EDTA-free, Boehringer Mannheim)].
- buffer A 20 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, ImM magnesium chloride, 10% glycerol, 20 mM imidazole
- buffer B [20 mM Tris-HCl buffer. (PH 8.5), 800 mM sodium chloride, ImM magnesium chloride, 10% glycerol, 20 mM imidazole]
- buffer C 20 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, ImM magnesium chloride, 10% glycerol, 100 mM imidazole.
- dialysis was performed against 300 ml of buffer D [20 mM Tris-HCl buffer (pH 8.5), 100 mM sodium chloride, ImM magnesium chloride, 10% glycerol].
- buffer E 20 mM Tris-HCl buffer ( ⁇ 8.5), 200 mM sodium chloride, ImM magnesium chloride, 10% dalycerol
- buffer F 20 mM Tris-HCl buffer ( ⁇ 8 5), 400 mM sodium chloride, ImM magnesium chloride, 10% glycerol
- buffer G 20 mM Tris-HCl buffer ( ⁇ 8.5), 800 mM sodium chloride, ImM magnesium chloride, 10% glycerol
- Example 11- (1) For the protein sample prepared in Example 11- (1) above, the dsRNA lig prepared in Example 2- (1), the protein sample prepared in Example 11- (1), l / il, 5X Reaction buffer (100 mM Tris-HCl buffer (pH 8.5), 750 mM sodium chloride, 12.5 mM magnesium chloride) 2 ⁇ l, add nuclease free water to make a total volume of 10 ⁇ l Liquid. After reaction at 37 ° C for 18 hours, 5 was subjected to 15% polyacrylamide gel electrophoresis and staining with ethidium bromide to confirm the cleavage product. As a result, a degradation product of about 21 base pairs was confirmed, and dsRNA degradation activity was confirmed.
- 5X Reaction buffer 100 mM Tris-HCl buffer (pH 8.5), 750 mM sodium chloride, 12.5 mM magnesium chloride
- Example 10_ (3) the protein sample (enzyme solution) 11 prepared in Example 11- (1), the CspB solution 1 ⁇ l prepared in Example 10- (2), Example 2—1 ⁇ g of dsRNA to be used as a substrate prepared in (1), 5X reaction buffer (100 mM Tris-HCl buffer (pH 8.5), 750 mM sodium chloride sodium salt, 12.5 mM sodium chloride salt magnesium) 21) 11 uclease free water was added to this to make a total amount of 10 / i1, which was used as a reaction solution.
- the CspB protein was added to a final concentration of 9 ⁇ 2 ⁇ ⁇ / ⁇ 1.
- a commercially available Dicer was also used as a control in this case.
- 2 ⁇ l of enzyme solution, 1 / i of CspB solution, 1 / ig of dsRNA as a substrate, 1 ⁇ l of 10 mM ATP solution, 0.5 mM of 50 mM magnesium chloride solution, 4 ⁇ l of the attached reaction buffer and nuclease-free water were added to the reaction buffer to make the volume 10 / i1, which was used as the reaction solution.
- the control water was obtained by adding nuclease free water instead of Cs pB.
- a mutant protein containing the ⁇ + RNaseIII domain of the present invention (hDi-ASI)
- CspB a mutant protein containing the ⁇ + RNaseIII domain of the present invention
- the amount of degradation of dsRNA by the addition of CspB was improved, and further, it was confirmed that almost no uncleaved substrate was present and almost all of the substrate was degraded.
- Dicer although an increase in dsRNA degradation was confirmed, it was confirmed that about half of the uncleaved substrate was present.
- Example 11 The RNA interference effect of the siRNA prepared using the protein of 11_ (1) was examined.
- a commercially available Dicer GTS
- Preparation of the dsRNA degradation product was basically performed by the method described in Example 11- (3) above.
- CspB prepared in Example 10- (2) was added to a final concentration of 9.2 ng / zl. That is, using the enzyme described in Example 11- (1) to which CspB was added and the commercially available Dicer, the dsRNA 10 / g portion prepared in Example 2- (1) was used at 37 ° C. Cut in 18 hours.
- These cleavage products were purified using Microcon-100 and Micropure-EZ (both manufactured by Takara Bio Inc.), and used for the following evaluation of RNA interference.
- a separate tube prepared by adding 47 ⁇ l of serum-free medium to 3 ⁇ l of Ribojuice Transfection Reagent (Takara Bio Inc.) was prepared and stirred vigorously. The mixture was allowed to stand at room temperature for 5 minutes, 55.6 ng of the above siRNA was added, and the mixture was gently mixed and allowed to stand at room temperature for 5 minutes.
- RNA interference occurs as the average fluorescence value decreases as compared to the control (vector only). Therefore, siRNA obtained by hDi-ASI + CspB showed an RNAi effect similarly to commercial Dicer, indicating that it is effective for RNAi. Furthermore, it was confirmed that siRNA could be produced efficiently, and that when the same amount of siRNA was used, a higher RNAi effect was obtained than with a commercially available enzyme.
- the present invention provides a protein having a dsRNA-degrading activity for producing a dsRNA of a specific length.
- the method of the present invention provides a method for promoting degradation into dsRNA of a specific length and / or a method for promoting RNA synthesis, which is useful in RNA interference and the like.
- a composition and a kit which can easily carry out the method for promoting the degradation of dsRNA of a specific length and / or the method for promoting RNA synthesis of the present invention.
- SEQ ID NO: 4 An amino acid sequence of human dicer mutant
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JP2005513163A JPWO2005017144A1 (ja) | 2003-08-14 | 2004-08-10 | dsRNA分解およびRNA合成方法 |
US10/567,731 US20070105113A1 (en) | 2003-08-14 | 2004-08-10 | Methods of degrading dsrna and synthesizing rna |
EP04771467A EP1652915A4 (en) | 2003-08-14 | 2004-08-10 | PROCESS FOR REMOVING DSRNA AND FOR SYNTHESIS OF RNA |
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JP2003-342126 | 2003-09-30 | ||
JP2003409639 | 2003-12-08 | ||
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JP (1) | JPWO2005017144A1 (ja) |
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WO2009117513A2 (en) * | 2008-03-21 | 2009-09-24 | The Regents Of The University Of California | Modified dicer polypeptide and methods of use thereof |
JP2011512821A (ja) * | 2008-02-29 | 2011-04-28 | ユニヴァーシティ オブ メディシン アンド デンティストリ オブ ニュージャーシィ | コールドショックタンパク質組成物、並びにその使用のための方法及びキット |
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JP5279339B2 (ja) * | 2008-05-16 | 2013-09-04 | タカラバイオ株式会社 | 逆転写反応用組成物 |
EP2393933A4 (en) * | 2009-02-04 | 2013-05-01 | Lucigen Corp | RNA AND DNA COPIERING ENZYMES |
Citations (2)
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WO1999027117A1 (fr) * | 1997-11-20 | 1999-06-03 | Takara Shuzo Co., Ltd. | Vecteur d'expression inductible a froid |
WO2001068836A2 (en) * | 2000-03-16 | 2001-09-20 | Genetica, Inc. | Methods and compositions for rna interference |
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WO2003093430A2 (en) * | 2002-05-03 | 2003-11-13 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for use in preparing sirnas |
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2004
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- 2004-08-10 WO PCT/JP2004/011480 patent/WO2005017144A1/ja active Application Filing
- 2004-08-10 JP JP2005513163A patent/JPWO2005017144A1/ja active Pending
- 2004-08-10 KR KR1020067003102A patent/KR20060098427A/ko not_active Application Discontinuation
- 2004-08-10 EP EP04771467A patent/EP1652915A4/en not_active Withdrawn
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WO1999027117A1 (fr) * | 1997-11-20 | 1999-06-03 | Takara Shuzo Co., Ltd. | Vecteur d'expression inductible a froid |
WO2001068836A2 (en) * | 2000-03-16 | 2001-09-20 | Genetica, Inc. | Methods and compositions for rna interference |
Non-Patent Citations (5)
Title |
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BAE W TE AL: "Escherichia coli CspA-family RNA chaperones are transcription antiterminators", PNAS, vol. 97, no. 14, 2000, pages 7784 - 7789, XP002904223 * |
KREMER W ET AL: "Solution NMR structure of the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima", EUR. J. BIOCHEM., vol. 268, 2001, pages 2527 - 2539, XP002904224 * |
MELEKHOVETS Y F ET AL: "Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro", NUCLEIC ACIDS RESEARCH, vol. 24, no. 10, 1996, pages 1908 - 1912, XP002904225 * |
PROVOST P ET AL: "Ribonuclease activity and RNA binding of recombinant human Dicer", THE AMBO JOURNAL, vol. 21, no. 21, 2002, pages 5864 - 5874, XP002904222 * |
See also references of EP1652915A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2011512821A (ja) * | 2008-02-29 | 2011-04-28 | ユニヴァーシティ オブ メディシン アンド デンティストリ オブ ニュージャーシィ | コールドショックタンパク質組成物、並びにその使用のための方法及びキット |
WO2009117513A2 (en) * | 2008-03-21 | 2009-09-24 | The Regents Of The University Of California | Modified dicer polypeptide and methods of use thereof |
WO2009117513A3 (en) * | 2008-03-21 | 2009-11-19 | The Regents Of The University Of California | Modified dicer polypeptide and methods of use thereof |
US8440430B2 (en) | 2008-03-21 | 2013-05-14 | The Regents Of The University Of California | Modified dicer polypeptide and methods of use thereof |
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EP1652915A1 (en) | 2006-05-03 |
JPWO2005017144A1 (ja) | 2007-10-04 |
KR20060098427A (ko) | 2006-09-18 |
US20070105113A1 (en) | 2007-05-10 |
TW200521237A (en) | 2005-07-01 |
EP1652915A4 (en) | 2006-08-30 |
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