JP2005229815A - Nuclear transferable nucleic acid structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technique for promoting the intranuclear localization and ensuring efficient transfection when a desired gene is transfected into a cell. <P>SOLUTION: An intranuclear localization nucleic acid structure is obtained by binding a nucleic acid substance containing a gene to be transfected into the interior of the nucleus of the cell to an importin protein (preferably an importin β) having functions to pass through nuclear membrane holes and related to intranuclear transportation through a covalently binding or noncovalently binding nonspecific interaction. A plasmid DNA is preferably used as the nucleic acid substance which is preferably bound to the importin protein by a biotin-avidin interaction. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a nucleic acid structure that functions as a non-viral vector that promotes cell nuclear translocation and improves transfection efficiency during transfection.

  Transfection that introduces a specific external gene into a cell is an indispensable means for obtaining useful information for disease treatment and drug development by analyzing the mechanism of action involved in the gene. Attempts have been made to use non-viral vectors for transfection to avoid adverse effects on the body. One of the major obstacles in improving the transfection efficiency of non-viral vectors is the translocation of external genes into the nucleus. To solve this problem, the use of nuclear protein translocation systems in eukaryotic cells has been actively addressed so far.

Nucleoproteins generally have a tag called nuclear translocation signal (NLS), and importin α as an intermediary with a transporter binds to the NLS peptide, and finally the import carrier β of import carrier β An original complex is formed and is transported in an energy-dependent and selective manner through the nuclear pores present in the nuclear membrane.
Gorlich, D., Vogel, F., Mills, AD, Hartmann, E., and Laskey, RA, Nature, 377, 246-248, (1995). Rexach, M., and Blobel, G., Cell 83,683-692, (1995). Yoneda, Y., J. Biochem. Tokyo 121,811-817 (1996).

So far, many studies have been made to promote nuclear translocation by conjugating an external gene and an NLS peptide, thereby improving the expression efficiency. However, there are cases where the effects of NLS have been confirmed, and there have been reports that a significant effect on expression efficiency has been denied, and no methodology has been established at present. One of the causes is the formation of a ternary complex, which is a two-step reaction, possibly reducing efficiency.
Zanta, MA, Belguise-Valladier, P., and Behr, JP, Proc. Natl. Acad. Sci. USA 96, 91-96 (1999). Collas, P., and Alestrom, P., Biochem. Cell. Biol., 75, 633-640 (1997). 11-506935 Special table 2002-514892 Special Table 2002-533088 Nagasaki, T., Myohoji, T., Tachibana, T., and Tamagaki, S., Bioconjugate Chem., 14, 282-286 (2003). Tanimoto, M., Kamiya, H., Minakawa, N., Matsuda, A., and Harashima, H., Bioconjugate Chem., 14, 1197-1202 (2003).

  An object of the present invention is to provide a new technique capable of facilitating nuclear translocation and ensuring efficient transfection when a desired gene is introduced into a cell.

Recently, the mechanism of translocation of substances that function in the nucleus does not require the formation of a ternary complex, and many substances (proteins) are formed by directly forming a complex with importin β, which is the main transport carrier. It has become clear that they are being transported within.
Jakel, S., and Gorlich, D., EMBOJ., 17, 4491 (1998). Truant, R., and Cullen, BR, Mol. Cell Biol., 19, 1210 (1999). Nagoshi, E., Imamoto, N., Sato, R., and Yoneda, Y., Mol. Biol. Cell, 10, 2221 (1999).

The present inventor has achieved the above object by paying attention to these phenomena and devising a means for binding the introduced gene to a specific intracellular factor involved in nuclear protein transport.
Thus, according to the present invention, a nucleic acid substance containing a gene to be introduced into the cell nucleus and an import protein that has a function of passing through the nuclear pore and is involved in nuclear transport are covalently or non-covalently bound. There is provided a nuclear translocating nucleic acid structure characterized by being bound via sex specific interactions.

  The nucleic acid structure of the present invention in which a transgene is combined with an importin protein, which is a transport factor, to form a complex (conjugate) is surely brought into the nucleus of the cell when brought into contact with a predetermined cell. Therefore, the gene can be efficiently introduced into the cell.

  In the present invention, an importin protein that binds to a nucleic acid substance containing a gene to be introduced into the nucleus of a cell to form a conjugate is a protein that has a function of passing through the nuclear pore and is involved in nuclear transport. Any of them can be used. Specifically, importin β, importin 7, transportin, transportin SR, CAS protein and the like can be mentioned, among which importin β is exemplified as a preferable one.

The amino acid sequences (base sequences) of these importin proteins and their actions can be known from, for example, the following documents.
Jakel, S., and Gorlich, D., EMBOJ., 17, 4491 (1998). Imamoto, N., Shimamoto, T., Kose, S., Takao, T., Tachibana, T., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y. FEBS Lett. 368, 415 (1995). Pollard, VW, Michael, WM, Nakielny, S., Siomi, MC, Wang, F., and Dreyfuss, G., Cell 86, 985 (1996). Nagoshi, E., Imamoto, N., Sato, R., and Yoneda, Y., Mol. Biol. Cell, 10, 2221 (1999). Kutay, U., Bischoff, FR, Kostka, S., Kraft, R., and Gorlich, D., Cell 90, 1061 (1997).

The binding between the nucleic acid substance containing a gene and the above-mentioned importin protein is carried out through a covalent or non-covalent specific interaction.
As a specific example of using a covalent bond, after introducing an amino group into a gene by chemical modification, using a bifunctional ε-maleimidocarboxylic acid active ester, a maleimide group is introduced, and further reacted with a cysteine residue of the import protein. And conjugating them to form conjugates. However, considering the conjugation of importin protein to many genes, the method by chemical modification of genes is difficult because it is a two-step reaction.

  On the other hand, when based on non-binding specific interaction, a reaction system that has been frequently used in the field of biochemistry can be used, which is preferable to the case of covalent bonding. Specific examples include GST protein-glutathione, histidine tag-nickel complex, biotin-avidin, and the like. If these reaction systems are used, for example, a nucleic acid substance containing a gene is modified with a GST protein, a histidine tag or biotin, and the importin protein is fused with glutathione, a nickel complex or avidin, so that The nucleic acid transferable nucleic acid structure of the present invention comprising a nucleic acid substance-importin protein conjugate (complex) is obtained through specific interaction.

Among the reaction systems exemplified above as those that exhibit non-covalent specific interaction, biotin-avidin having a particularly large binding force is preferable. Kits for the biotinylation of nucleic acids are also commercially available and can be easily modified, and nucleic acid substances containing various genes can be easily biotinylated. In general, streptavidin is preferably used as avidin. If streptavidin fusion importin protein is used, it is due to the highly stable non-covalent specific interaction (dissociation constant: <10 ^-15 ) of biotin-streptavidin. It is possible to easily obtain the nucleic acid structure of the present invention comprising a conjugate obtained by binding an importin protein to a nucleic acid substance containing various genes.

  In the present invention, the nucleic acid substance is a gene itself to be introduced into a cell or a nucleic acid containing the gene, and can be in any form as long as it is combined with importin by the means described above. Well, RNA, oligo DNA, single-stranded nucleic acid, double-stranded nucleic acid, plasmid DNA and the like are included, but a particularly suitable nucleic acid substance from a practical viewpoint is plasmid DNA. Here, plasmid DNA is used to mean an expression vector, that is, one that encodes a protein to be expressed downstream of a promoter, and its specific base sequence varies depending on the target protein.

  Thus, a particularly preferred embodiment of the nuclear translocation nucleic acid structure of the present invention is that a nucleic acid substance containing a gene to be introduced into a cell is plasmid DNA, the plasmid DNA is labeled with biotin, and a streptavidin fusion import It is bound to a protein (preferably importin β).

  In such a case, as the biotin (labeled) plasmid, it is preferable to use a compound having a polyethylene glycol chain (PEG) having a molecular weight of 3000 to 5000 that gives a sufficient distance between the plasmid DNA and biotin. In addition, biotinylation of plasmid DNA is performed by chemical modification by diazo coupling, but the number of biotin introduced per molecule is preferably 5 to 20, and more preferably 5 to 10.

  The nuclear translocation nucleic acid structure of the present invention is used for injecting a specific gene into a cell, cloning the gene, or expressing a protein encoded by the gene by contacting with a predetermined cell. However, the target cells are not limited and the principles of the present invention can be applied to any kind of eukaryotic cells, especially animal cells. However, it is preferable that the importin protein used has the same origin as the target cell type.

  Hereinafter, in order to more specifically show the characteristics of the present invention, it relates to the preparation of plasmid DNA-importin protein conjugate, which is a nucleic acid structure according to the present invention, and evaluation of promotion of nuclear translocation and protein expression in in vitro tests using the same. Although an Example is described, this invention is not limited by these Examples.

Preparation of biotin-labeled plasmid pDsRed (plasmid DNA encoding red fluorescent protein, 4700 bp, manufactured by Clontech) was used as plasmid DNA. First, 5.17 mg of a biotin-modified aniline derivative having a polyethylene glycol chain synthesized according to the scheme shown in FIG. 1 ((I) in FIG. 1, hereinafter referred to as biotin-PEG-modified aniline derivative) was cooled to 4 ° C. 53 mg of 10 mg / ml NaNO ₂ dissolved in M HCl (116 μL) and cooled to 4 ° C. with good stirring was added and stirred at 4 ° C. for 5 minutes.
Thereafter, 1M NaOH (57.8 μL) was added to complete the reaction. This solution was added to 752 μL of 0.13 μg / μL pDsRed (in 0.1 M borate buffer (pH = 9.0)) and stirred at room temperature for 15 minutes. It was placed on a molecular sieve filter of Ultrafree-MC (MW: 10000) and centrifuged at 14000 g at 4 ° C. to remove unreacted biotin-PEG-modified aniline derivative. Further, 400 μL of 70% ethanol was added and washed by centrifugation at 14,000 g at 4 ° C. After drying, the synthesized biotin-labeled plasmid ((II) in FIG. 1, hereinafter referred to as biotin-PEG-pDsRed) (91 μg) (91 μg) was dissolved in 60 μL of PBS (−) and stored at −20 ° C.

Biotin -PEG-pDsRed (2μg), Texas Red labeled streptavidin (texas)
red labeled streptavidin) (TR-S, 5 μg) was mixed in PBS (20 μL) to prepare a conjugate. Texas Red labeled streptavidin was released using ultrafiltration membrane (Microcon YM-100, NMWL: 100,000) by adding 1.5M NaI (200 μL) to dissociate non-specifically bound Texas Red labeled streptavidin Was removed. After washing with PBS (200 μL), Texas Red labeled pDsRed (hereinafter referred to as pDsRed-biotin- (TR-S) ₄ ) was obtained.
Next, to determine the DNA concentration, add 5 μL of 1 mg / mL Hoechst solution to PBS (2 mL), and gradually add pDsRed-biotin- (TR-S) ₄ solution to 5, 10, 15, 20 μL. The fluorescence intensity of the added Hoechst was measured. At this time, a calibration curve of Hoechst fluorescence intensity was prepared with a DNA solution having a known concentration, and the DNA concentration of the pDsRed-biotin- (TR-S) ₄ solution was determined. On the other hand, based on the calibration curve of the fluorescence intensity and concentration of TR-S, gradually add pDsRed-biotin- (TR-S) ₄ solution to PBS (2 mL) to 5, 10, 15, 20 μL, The concentration of Texas Red was determined. The number of biotin labels per plasmid DNA was determined to be 9.4, calculated from this DNA concentration and Texas Red concentration.

Streptavidin fusion importin beta proteins regulating made importin beta (Importinbeta) and GFP (green fluorescent protein) coding for pGEX2T-GFP-impβ plasmid vector [15] of importin region EcoRI and HindIII encoding beta A streptavidin gene having the same restriction enzyme site was inserted into the site to construct a GFP-importin β-streptavidin fusion protein (hereinafter referred to as GβS) expression vector pGβS (SEQ ID NO: 1) shown in FIG. Importin β encoded by pGEX2T-GFP-impβ is derived from a mouse.
The constructed pGβS was transformed into E. coli BL21 and cultured in LB medium to obtain a recombinant fusion protein GβS. GβS is GST protein (26K Da), GFP (27K Da), 5 '643base importin β (76K Da, total length 97K Da), streptavidin (16K) It is a recombinant protein containing a fragment of Da) and has a molecular weight of 145K Da. The results of SDS-PAGE of this recombinant GβS protein are shown in FIG. A single band is shown at the corresponding molecular weight, indicating that the GβS protein was successfully expressed and purified.
Kose, S., Imamoto, N., Shimamoto, T., Tachibana, T., Shimamoto, T., and Yoneda, YJ Cell Biol. 139, 841 (1997).

Evaluation of nuclear translocation ability by microinjection method After injection into the cytoplasm by microinjection method, after culturing for a certain period of time, the cells were fixed with 5% HCHO solution, and the intracellular kinetics of the injected substance was observed. If it has the ability to enter the nucleus, the nucleus is stained. Using biotin-PEG-pDsRed (9.4) (number of biotin introduced per plasmid: 9.4) prepared by the method of Example 1, 2.0 μg / μL biotin-PEG-pDsRed (9.4) PBS solution (30 μL) and 2.4 μg / μLGβS PBS solution (30 μL) was mixed and immediately microinjected into the cytoplasm of NIH3T3 cells. FIG. 4 (a) shows that the green fluorescence of GFP was observed after 1 hour of fixation (shown in monochrome for convenience). In addition, after blocking nonspecific adsorption of protein with skim milk, Texas Red labeled streptavidin was added to the cells, the binding treatment to biotin was performed, and the fluorescence of Texas Red was observed. , Shown in monochrome).
Based on the above results, the fusion protein and biotin-labeled plasmid microinjected into the cytoplasm in NIH3T3 cells are localized in the nucleus, facilitating nuclear translocation of biotin-labeled plasmid DNA and streptavidin-importin β fusion. It became clear that

Protein expression experiment by microinjection Recombinant fusion protein GβS promotes translocation of biotin-labeled plasmid DNA into the nucleus, so that it can be expected to be efficiently transcribed in the nucleus after injection into the cytoplasm and enhance the protein expression level. . Therefore, in order to achieve a plasmid concentration of 0.5 μg / μL, and when complexing with the fusion protein GβS, the final concentration of the fusion protein GβS is 0.6 μg / μL. After microinjection, the percentage of cells in which the expression of red fluorescent protein was confirmed after 24 hours was examined.
The result is shown in FIG. No protein expression was observed when microinjection was performed with biotin chemically modified plasmid DNA alone (at the bottom in FIG. 5). On the other hand, the nucleic acid structure of the present invention comprising a plasmid DNA-importin β conjugate (the middle part in FIG. 5) has improved expression efficiency compared to an intact plasmid DNA (the uppermost part in FIG. 5). Thus, it can be seen that the plasmid DNA-importin β conjugate enhances protein expression by promoting nuclear transfer of plasmid DNA.

  The present invention is expected to be used as a new non-viral technique for introducing a desired gene into cells in various fields including gene therapy.

A biotin-labeled plasmid DNA synthesis scheme used to prepare the nucleic acid structure of the present invention is shown (Example 1). (Example 2) which shows the vector map of the recombinant fusion protein G (beta) S expression vector used for producing the nucleic acid structure of this invention. The confirmation by SDS-PAGE of GβS protein expression is shown (Example 2). (Example 3) which shows the fluorescence observation result by the microinjection method to the NIH3T3 cell for evaluating the nuclear translocation ability of the nucleic acid structure of this invention. (Example 4) which shows the expression efficiency after the microinjection in the protein expression experiment conducted about the nucleic acid structure and comparative sample of this invention.

Claims (8)

  A nucleic acid substance containing a gene to be introduced into the nucleus of a cell and an importin protein that has a function of passing through the nuclear pore and is involved in nuclear transport through a covalent or non-covalent specific interaction. A nuclear translocating nucleic acid structure characterized by being bound to each other.   The nuclear importable nucleic acid structure according to claim 1, wherein the importin protein is a protein selected from importin β, importin 7, transportin, transportin SR, or CAS protein. .   The importable nucleic acid structure according to claim 2, wherein the importin protein is importin β.   The nuclear translocation nucleic acid structure according to any one of claims 1 to 3, wherein the nucleic acid substance and the importin protein are bound to each other via a non-covalent specific interaction.   The nuclear translocation nucleic acid structure according to claim 4, wherein the non-covalent specific interaction is a biotin-avidin interaction.   6. The nuclear translocating nucleic acid structure according to any one of claims 1 to 5, wherein the nucleic acid substance is plasmid DNA.   The nuclear transferable nucleic acid structure according to claim 6, wherein the plasmid DNA is labeled with biotin and bound to a streptavidin fusion importin protein. A method for introducing a gene into a cell, comprising the step of bringing the nuclear translocation nucleic acid structure according to any one of claims 1 to 7 into contact with the cell.