WO2017047750A1 - Dna for intracellular penetration, and method for introducing target molecule into cell using said dna - Google Patents

Abstract

The present invention provides: DNA for intracellular penetration; a composition that includes said DNA and a target molecule to be introduced into a cell; a method for introducing the target molecule into the cell using said composition; and an agent and kit for introducing the target molecule into the cell, said agent and kit including said DNA. Provided is DNA for intracellular penetration, said DNA including an intracellular penetration region comprising the following base sequence: 5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-N22-N23-TCG-N27-N28-N29-G-N31-AT-N34-N35-GTG-N39-N40-TCG-3' [Herein, N1 is deleted or is T, N2 is G or is deleted, N5 is C or T, N6 is G or C, N7 is G or T, N14 is G or C, N19 is A or G, N20 is G or A, N22 is deleted or is T, N23 is T or A, N27 is G, T, or A, N28 is T, A, or C, N29 is deleted or is T, N31 is G or A, N34 is G or is deleted, N35 is C or A, N39 is G or T, and N40 is G or A] (SEQ ID NO: 1).

Description

Intracellular DNA and method for introducing a target molecule into the cell using the same

The present invention relates to intracellular DNA. The present invention also relates to a composition comprising the DNA and a target molecule to be introduced into a cell, and a method for introducing the target molecule into the cell using the composition. Furthermore, the present invention relates to an intracellular introduction agent of a target molecule and a kit for introduction into the cell, which contain the DNA.

Techniques for introducing molecules such as nucleic acids, peptides or proteins into cells are important for various purposes including elucidation of the function of these molecules or treatment of disease or pathogenesis. However, in general, molecules such as proteins and nucleic acids are highly hydrophilic and usually hardly permeate cell membranes. For this reason, many techniques for introducing these molecules into cells have been developed.

For example, technologies for introducing nucleic acids into cells include electroporation methods that electrically open holes in cell membranes and incorporate nucleic acids into cells, microinjection methods that directly inject nucleic acids through glass capillaries, and liposomes that incorporate nucleic acids. And a lipofection method using a virus, and a virus vector method using a virus such as a retrovirus. However, the electroporation method and the microinjection method physically damage the cells and introduce nucleic acids or the like, so that damage to the cells is large and a dedicated device is required. In the electroporation method, it is necessary to examine introduction conditions according to the cells, which is troublesome. In the lipofection method, the toxicity of the reagent to the cells becomes a problem, and the cost is high. The virus vector method has a problem that it takes time to prepare a vector, and a nucleic acid or protein other than the target may be introduced. In other words, currently available technologies have many problems in terms of introduction efficiency, cytotoxicity, cost, labor, safety, and the like.

In addition, nucleic acid drugs that have been rapidly developed in recent years are generally difficult to introduce into the living body, and an efficient drug delivery system needs to be developed.

There is also known a method for introducing a molecule such as a protein or nucleic acid into a cell using a peptide having a property of moving into a cell, commonly called a cell membrane permeable peptide (CPP). Representative cell membrane-penetrating peptides include Tat derived from HIV-1 virus and penetratin derived from Drosophila (Patent Documents 1 and 2).

Similarly, RNA having the property of migrating into cells has been reported (Non-patent Document 1 and Patent Document 3). This RNA is selected from RNA libraries containing random sequences by selecting the RNA that has migrated into the cell and amplifying it multiple times to select only the RNA that migrates into the cell (referred to as the Cell-SELEX method). ). However, since RNA is generally easily degraded, there are many problems such as stability in order to put into practical use a technique for introducing other molecules into cells using this intracellularly transferred RNA.

Japanese Patent Laid-Open No. 10-33186 (International Publication No. 94/04686) JP-T 2002-530059 Publication (International Publication No.00 / 29427) International Publication No. 2008/124798

The present invention includes a cell translocating DNA, a composition comprising the DNA and a target molecule to be introduced into the cell, a method for introducing the target molecule into the cell using the composition, and the DNA. It is an object of the present invention to provide an intracellular introduction agent of a target molecule and a kit for intracellular introduction.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found a DNA having the ability to move into cells and have completed the present invention.

That is, the present invention includes the following.
[1] The following base sequence:
5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-N22-N23-TCG-N27-N28-N29-G-N31-AT-N34-N35-GTG -N39-N40-TCG-3 '
[here,
N1 is deleted or T,
N2 is G or is deleted;
N5 is C or T;
N6 is G or C;
N7 is G or T,
N14 is G or C;
N19 is A or G;
N20 is G or A,
N22 is deleted or T,
N23 is T or A,
N27 is G, T or A,
N28 is T, A or C;
N29 is deleted or T,
N31 is G or A;
N34 is G or is deleted;
N35 is C or A,
N39 is G or T, and N40 is G or A] (SEQ ID NO: 1)
Intracellular DNA comprising an intracellular translocation region consisting of
[2] The intracellular DNA according to [1], wherein the base sequence is the following (a) or (b).
(a) 5'-GGGCGGGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 2),
5'-GGTGGGGTGGTGGGGGAGGTTCGTTGGATGCGTGGGTCG-3 '(SEQ ID NO: 3),
5'-GGGCGGGGTGGTCGGGGGAGTTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 4),
5'-GGGCGGGGTGGTGGGGGAGGTTCGTATGGATGCGTGGATCG-3 '(SEQ ID NO: 5),
5'-GGGCGGGGTGGTGGGGGAGGTTCGATGGATCGTGGGTCG-3 '(SEQ ID NO: 6),
5'-GGGCGGGGTGGTGGGGGAGGATCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 7),
5'-GGCGGGGTGGTGGGGGAGGTTCGATGGATGAGTGGGTCG-3 '(SEQ ID NO: 8),
5'-GGGCCGGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 9),
5'-GGGCGGGGTGGTGGGGGAGGTTCGACGGATGCGTGGGTCG-3 '(SEQ ID NO: 10),
5'-TGGGCGGGGTGGTGGGGGAGGTTCGGTGAATGCGTGTGTCG-3 '(SEQ ID NO: 11), and 5'-GGGCGTGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3' (SEQ ID NO: 12)
(B) a base sequence in which one or two bases are substituted, deleted, inserted or added in the base sequence shown in any of SEQ ID NOs: 2 to 12
[3] The following base sequence:
5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-3 '
[here,
N1 is deleted or T,
N2 is G or is deleted;
N5 is C or T;
N6 is G or C;
N7 is G or T,
N14 is G or C;
N19 is A or G, and N20 is G or A] (SEQ ID NO: 13)
Intracellular DNA comprising an intracellular translocation region containing 2 or more of.
[4] Intracellular DNA according to [3], wherein each of the base sequences is the following (a) or (b):
(a) 5'-GGGCGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 14),
5'-GGTGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 15),
5'-GGGCGGGGTGGTCGGGGGAG-3 '(SEQ ID NO: 16),
5'-GGCGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 17),
5'-GGGCCGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 18),
5'-TGGGCGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 19), and 5'-GGGCGTGGTGGTGGGGGAGG-3' (SEQ ID NO: 20)
(B) a base sequence in which one base is substituted, deleted, inserted or added in the base sequence shown in any of SEQ ID NOS: 14 to 20
[5] A multimer of intracellular translocating DNA formed by binding two or more intracellular translocating DNAs, each of the intracellular translocating DNA being any one of [1] to [4] Intracellular DNA multimer selected from the intracellular translocation DNA described in 1.
[6] An agent for introducing a target molecule into the cell, which comprises the intracellular transferable DNA according to any one of [1] to [4] or the intracellular transferable DNA multimer according to [5].
[7] Intracellular DNA according to any one of [1] to [4] or intracellular multimer DNA according to [5], and a target molecule to be introduced into the cell, Composition.
[8] A method for introducing a target molecule into a cell, comprising contacting the composition according to [7] with a cell.
[9] A kit for introducing a molecule of interest into a cell, comprising the intracellular translocation DNA according to any one of [1] to [4] or the intracellular translocation DNA multimer according to [5].

This specification includes the disclosure of Japanese Patent Application No. 2015-185295, which is the basis of the priority of the present application.

According to the present invention, a cell-introducing DNA, a composition comprising the DNA and a target molecule to be introduced into the cell, a method for introducing the target molecule into the cell using the composition, and the DNA, Intracellular introduction agents of target molecules and kits for intracellular introduction are provided.

It is a schematic diagram which shows the exemplary structure of a cell transferable DNA multimer. (A) shows the structure of a unit in which intracellular DNA is directly bound to the scaffold strand, (B) shows indirect binding of intracellular DNA to the scaffold strand (via the adapter strand) The structure of the unit is shown. (C) and (D) show the structures of the trimers of the units shown in (A) and (B), respectively. 2 is a schematic diagram showing the structure of a test DNA used in Example 2. FIG. (A) shows the structure of the test DNA monomer (QAp1 monomer), and (B) shows the structure of the test DNA trimer (QAp1 trimer as an example). It is a figure which shows the flow cytometry result of HepG2 cell (A) or 3T3-L1 cell (B) incubated with the QAp1 monomer. It is a figure which shows the flow cytometry result of 3T3-L1 cell which incubated with the QAp1 monomer, and was treated with trypsin and DNase. Incubated with QAp1 trimer (A), QAp3 trimer (B), QAp4 trimer (C), QAp7 trimer (D), QAp8 trimer (E) or QAp10 trimer (F) It is a figure which shows the flow cytometry result of HB2 cell. Flow cytometry results of HB2 cells incubated with QAp11 trimer (G), QAp14 trimer (H), QAp15 trimer (I), QAp16 trimer (J) or QAp19 trimer (K) FIG. It is a figure which shows the flow cytometry result of HEK293T cell (A) or HW cell (B) incubated with QAp1 trimer. It is a figure which shows the flow cytometry result of HEK293T cell (A), HB2 cell (B), or HW cell (C) which was incubated with QAp1 trimer, and was treated with trypsin and DNase. It is a figure which shows the flow cytometry result of MCF-7 cell which incubated with the QAp1A monomer (A) or the QAp1AA monomer (B), and was treated with trypsin and DNase.

Hereinafter, the present invention will be described in detail.
<Intracellular DNA>
The present invention relates to intracellular DNA. In the present specification, intracellular translocation DNA means DNA having the ability to translocate into cells.

In this specification, “intracellularity” or “intracellularity” refers to the ability to migrate from the outside of the cell to the inside of the cell membrane separating the inside and outside of the cell. The inner side of the cell membrane includes the cytosol and any compartment within the cell including, for example, endosomes and vesicles. The transition pathway is not particularly limited, and includes pathways through transport proteins and pathways such as endocytosis, phagocytosis, and pinocytosis. For example, the intracellular DNA of the present invention may first bind to the cell surface and then be taken into the cell by endocytosis.

Whether or not DNA has the ability to move into cells can be appropriately determined by those skilled in the art. For example, it can be determined by adding test DNA bound to a fluorescent substance to cells, incubating, decomposing DNA bound to the cell surface, and then measuring the fluorescence intensity of the cells with a flow cytometer. Alternatively, it can be determined, for example, by adding test DNA bound to a fluorescent substance to cells and incubating them, and then observing fluorescence inside the cells with a confocal laser microscope. For example, when the fluorescence intensity of a cell (for example, HepG2 cell, HB2 cell, HW cell or HEK293T cell) that has been added and incubated with a test DNA conjugated with a fluorescent material (for example, FITC) is measured with a flow cytometer, the fluorescent material The average fluorescence intensity per cell is larger than the control DNA bound to (for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more or 2.5 times or more) and / or when the value of the fluorescence intensity showing the peak of the cell count increases, It can be determined that DNA has the ability to translocate into cells. As the control DNA, a double-stranded DNA molecule obtained by binding (annealing) a test DNA with a complementary DNA can be used.

The intracellular transferable DNA of the present invention includes an intracellular transfer region. In the present specification, the intracellular translocation region is a region involved in the intracellular translocation ability of DNA containing the region.

In one embodiment, the intracellular translocation DNA of the present invention comprises
The following nucleotide sequence: 5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-N22-N23-TCG-N27-N28-N29-G-N31-AT- N34-N35-GTG-N39-N40-TCG-3 '
[Where N1 is deleted or T, N2 is G or deleted, N5 is C or T, N6 is G or C, N7 is G or T N14 is G or C, N19 is A or G, N20 is G or A, N22 is missing or T, N23 is T or A, and N27 is G , T or A, N28 is T, A or C, N29 is deleted or T, N31 is G or A, N34 is G or is deleted, N35 is C or A, N39 is G or T, and N40 is G or A] (SEQ ID NO: 1)
An intracellular translocation region consisting of The base sequence is preferably 5'-GGGCGGGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 2), 5'-GGTGGGGTGGTGGGGGAGGTTCGTTGGATGCGTGGGTCG-3' (SEQ ID NO: 3), 5'-GGGCGGGGTGGTCGGGGGAGTTTCGGTGGATGCGTGGGTCG-3 ' GGGCGGGGTGGTGGGGGAGGTTCGTATGGATGCGTGGATCG-3 '(SEQ ID NO: 5), 5'-GGGCGGGGTGGTGGGGGAGGTTCGATGGATCGTGGGTCG-3' (SEQ ID NO: 6), 5'-GGGCGGGGTGGTGGGGGAGGATCGGTGGATGCGTGGGTGT, GG '-GGGCCGGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3' (SEQ ID NO: 9), 5'-GGGCGGGGTGGTGGGGGAGGTTCGACGGATGCGTGGGTCG-3 '(SEQ ID NO: 10), 5'-TGGGCGGGGTGGTGGGGGAGGTTCGGTGAATGCGTGTGGCG' ). Alternatively, the base sequence may be a base sequence in which one or two bases are substituted, deleted, inserted or added in the base sequence shown in any of SEQ ID NOs: 2 to 12. In general, when a nucleic acid having a desired property (e.g., an aptamer capable of binding to a specific molecule) is obtained, the nucleotide sequence can be changed by substituting, deleting, inserting or adding several nucleotide sequences. Can be optimized. Thus, in the base sequence shown in any one of SEQ ID NOs: 2 to 12, DNA containing a base sequence optimized by substitution, deletion, insertion or addition of one or two bases is also available for cells. It can have internal migration ability.

In another embodiment, the intracellular translocation DNA of the present invention comprises
The following nucleotide sequence: 5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-3 '
[Where N1 is deleted or T, N2 is G or deleted, N5 is C or T, N6 is G or C, N7 is G or T N14 is G or C, N19 is A or G, and N20 is G or A] (SEQ ID NO: 13)
May contain an intracellular translocation region. Each of the base sequences is preferably 5′-GGGCGGGGTGGTGGGGGAGG-3 ′ (SEQ ID NO: 14), 5′-GGTGGGGTGGTGGGGGAGG-3 ′ (SEQ ID NO: 15), 5′-GGGCGGGGTGGTCGGGGGAG-3 ′ (SEQ ID NO: 16), 5 '-GGCGGGGTGGTGGGGGAGG-3' (SEQ ID NO: 17), 5'-GGGCCGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 18), 5'-TGGGCGGGGTGGTGGGGGAGG-3' (SEQ ID NO: 19), and 5'-GGGCGTGGTGGTGGGGGAGG-3 '(SEQ ID NO: 20 ). Alternatively, each of the base sequences may be a base sequence in which one base is substituted, deleted, inserted or added in the base sequence shown in any of SEQ ID NOs: 14 to 20. The two or more base sequences may be, for example, 2 to 10, 2 to 5, or 2 to 3. Each of the two or more base sequences may be the same or different. For example, the intracellular translocation region includes two of the base sequences, and the two base sequences may both be the base sequence shown in SEQ ID NO: 14, or the base sequence and the sequence shown in SEQ ID NO: 14. The base sequence indicated by number 15 may be used. The base sequences contained in the intracellular translocation region may be adjacent to each other or any other base sequence having a length of 1 to 10, 1 to 5, or 1 to 3 bases (for example, T, TT , Or TTT).

The intracellular transferable DNA of the present invention may contain any other nucleotide sequence in addition to the intracellular transfer region. Intracellular DNA of the present invention is, for example, a base sequence involved in the multimerization of DNA described later (multimerization region), a base sequence involved in binding to the target molecule described later, such as a spacer sequence ( For example, via T, TT, or TTT).

The length of the intracellular DNA of the present invention is not particularly limited, but is 38 to 400 bases, 38 to 300 bases, 39 to 200 bases, 39 to 100 bases, or 39 to 50 bases long. It's okay.

Intracellular DNA of the present invention is a protective group, functional group at the phosphate group, sugar and / or base moiety, or at the 3 'end and / or 5' end, as long as the characteristics of the present invention are not impaired Alternatively, it may be appropriately modified with a substituent (for example, methylation, halogenation) or a labeling substance (for example, a radioisotope such as ³² P, ³ H, ¹⁴ C, ¹³ C, or FITC, DIG, or biotin). . Further, in the intracellular DNA of the present invention, the oxygen atom of the phosphate group in the nucleotide constituting the DNA may be substituted (phosphorothioated) with a sulfur atom.

The intracellular migration DNA of the present invention can be produced by a conventional method well known in the art. For example, it can be produced by manual or automated reaction, enzymatically or by chemical synthesis. When chemically synthesizing DNA molecules, a contract manufacturing service of a manufacturer (for example, Thermo Fisher Scientific, Greiner Japan, Sigma Aldrich, etc.) may be used. The synthesized DNA molecules may be purified from the mixture, for example, by extraction with a solvent or resin, precipitation, electrophoresis or chromatography.

<Multimer>
Intracellular DNA of the present invention may be multimerized by binding two or more. Each of the intracellular DNA contained in the multimer may be the same or different. A multimer in which two or more intracellular transferable DNAs of the present invention are bound is preferable because it has higher intracellular transferability than a single intracellular transferable DNA (monomer). The number of intracellular translocating DNAs of the present invention bound in the multimer is not particularly limited, but may be 2 to 5, 2 to 4, or 2 to 3. For example, the multimer may be a dimer or a trimer.

The multimer may be one in which intracellular DNAs are directly bound to each other, or indirectly (for example, via a linker molecule or the intracellular region contained in the intracellular DNA of the present invention). Other than the base sequence (multimerization region) other than the nucleic acid chain (adapter chain) having a complementary base sequence). Alternatively, the multimer may be one in which the intracellular transferable DNAs of the present invention are bound to each other using a biotin-avidin bond (see, for example, JP 2010-158237 A).

For example, a multimer may be formed by combining two or more units, each unit containing the intracellularly transferred DNA of the present invention and a scaffold chain. An exemplary structure of the unit is shown in FIG. 1A. The scaffold strand may be any nucleic acid strand. Intracellular DNA may include an intracellular translocation region and a multimerization region involved in multimerization. The multimerization region can bind to the scaffold strand through complementary base pairing. The binding between the multimerization region and the scaffold chain may be direct (e.g., as illustrated in FIG.1A) or indirect (e.g., via the adapter chain) (e.g. As illustrated in FIG. 1B). Two or more units can be linked directly or indirectly (eg, via a spacer sequence such as T, TT, or TTT) by binding the scaffold chains together. For example, FIG. 1C shows the structure of a trimer that is a multimer in which three units shown in FIG. 1A are linked via a spacer sequence. FIG. 1D shows the structure of a trimer, which is a multimer in which three units shown in FIG. 1B are linked via a spacer sequence.

In the present invention, the intracellular migration region contained in the intracellular migration DNA is preferably single-stranded. When the intracellular translocation region is annealed with a nucleic acid containing a complementary or substantially complementary base sequence to become double-stranded, the intracellular translocation ability of the intracellular translocation DNA of the present invention is reduced or ineffective. Because it becomes.

The intracellular translocation DNA or multimer thereof of the present invention not only can itself migrate into cells, but also has the property of transferring other molecules bound to the DNA or multimers into cells. Therefore, the intracellular translocation DNA or multimer thereof of the present invention includes a composition for introducing a target molecule into a cell, a method for introducing the target molecule into a cell, and an agent for introducing the target molecule into the cell and the cell. It is useful in a kit for internal introduction.

<Composition>
There is also provided a composition comprising the intracellularly transferred DNA of the present invention or a multimer thereof and a target molecule to be introduced into the cell. The composition of the present invention can be used for introducing a target molecule into cells in vitro or in vivo. The composition of the present invention may be a pharmaceutical composition.

In the present specification, the target molecule is not limited, for example, nucleic acid, peptide or protein (for example, hormone, growth factor, enzyme, toxin, antibody or antibody fragment, etc.), lipid, sugar, drug (for example, antitumor drug) As well as other synthetic or natural compounds (eg, labeled substances such as FITC, biotin or Cy3). The target molecule may have a functional group selected from an azide group and an alkyne group. The target molecule may preferably be a nucleic acid, peptide, or protein that is poor in cell internalization.

The target molecule is more preferably a nucleic acid. Examples of the nucleic acid include, but are not limited to, single-stranded or double-stranded DNA, RNA, DNA-RNA chimeric nucleic acid, and the like. Nucleic acids that can be used as target molecules can be miRNAs, siRNAs, antisense nucleic acids, decoy nucleic acids, aptamers, miRNA inhibitors or gapmers. Alternatively, the nucleic acid that can be used as the target molecule can be double-stranded DNA containing an expression cassette (for example, plasmid DNA or double-stranded DNA obtained by linearizing plasmid DNA). Typically, an expression cassette can include at least a promoter sequence and a gene coding sequence operably linked thereto.

The size of the nucleic acid that can be used as the target molecule is not limited. For example, in the case of a high molecular nucleic acid such as plasmid DNA, it may be 2,000 to 10,000 bases, 2,000 to 8,000 bases, or 2,000 to 5,000 bases. The size of a nucleic acid that can be used as a target molecule is, for example, 5 to 1,000 bases, 10 to 500 bases, 10 to 300 bases, 10 to 100 bases, or 20 to 30 bases in the case of a low molecular weight nucleic acid such as siRNA. It may be.

As used herein, the cells may be derived from any organism, but eukaryotes such as vertebrates such as mammals (e.g., humans, monkeys, cows, mice, rats), birds, amphibians, It may be a cell derived from a microorganism such as a fish, a plant, or a yeast. The cell may be a cultured cell line such as a cancer cell, or a primary cultured cell isolated from an individual or tissue. Examples of cultured cell lines include HEK293T cells, HeLa cells, 3T3-L1 cells, HepG2 cells, HB2 cells, HW cells, and MCF-7 cells. The cultured cell lines may be HB2 cells and HW cells that have low gene transfer efficiency with commercially available transfection agents. The cells may be stem cells such as pluripotent stem cells (eg, ES cells and iPS cells) or mesenchymal stem cells. The cells may be hepatocytes, kidney cells, cervical cells, adipose precursor cells, brown adipose precursor cells, white adipose precursor cells, or mammary cells, or cells derived from these cells. Further, the cell may be not only a cultured cell or an isolated cell but also a cell in a tissue (preferably taken out of a living body) or in an individual.

The composition of the present invention can also be used in vivo by administering to a subject. As used herein, a subject can include a eukaryote, such as a vertebrate, plant, etc. as described above.

In the composition of the present invention, the target molecule may or may not be bound to intracellular migration DNA or a multimer thereof. Even if the target molecule is not bound to the intracellular DNA or multimer thereof at the time of administration of the composition, the target molecule is mixed and bound with the intracellular DNA or multimer after administration to bind intracellularly. The target molecule can be introduced into the cell by the action of the migratory DNA.

In the present specification, the binding between the target molecule and the intracellular translocation DNA of the present invention or a multimer thereof can maintain the binding state in which the target molecule can be introduced into the cell by the action of the intracellular translocation DNA. Any binding mode may be used. Examples of the bonding mode include a covalent bond or a non-covalent bond such as a hydrogen bond, an ionic bond, and a van der Waals bond. The binding of the target molecule to the intracellular DNA of the present invention or a multimer thereof may be direct or indirect (for example, via a linker molecule or spacer molecule, or into the intracellular cell of the present invention). Or a nucleic acid chain (adapter chain) having a base sequence complementary to an arbitrary base sequence other than the intracellular translocation region contained in the sex DNA.

The target molecule may bind to any position of the intracellular transferable DNA of the present invention or a multimer thereof, for example, may bind to the 5 ′ side and / or 3 ′ side of the intracellular transferable DNA. .

When the target molecule is a single-stranded nucleic acid, the single-stranded nucleic acid may be present on the same nucleic acid chain as the intracellular DNA of the present invention. When the target molecule is a double-stranded nucleic acid, one strand of the double-stranded nucleic acid may be present on the same nucleic acid strand as the intracellular DNA of the present invention.

The number of target molecules that bind to the intracellular DNA or multimer thereof of the present invention is not particularly limited, but may be one or more, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. . When the number of target molecules that bind to the intracellular DNA or multimer thereof of the present invention is 2 or more, each target molecule may be the same or different. Further, the number of intracellularly transferred DNAs or multimers thereof of the present invention that bind to the target molecule is not particularly limited, but may be one or more, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. possible. When the number of intracellularly transferred DNAs or multimers thereof of the present invention that bind to the target molecule is 2 or more, each intracellularly transferred DNA or multimer thereof may be the same or different.

The ratio (molar ratio) between the intracellular translocating DNA of the present invention or its multimer and the target molecule in the composition is not particularly limited, but is 1:10 to 10: 1, 1: 5 to 5: 1, Or from 1: 2 to 2: 1, for example 1: 1.

In the state where the intracellular transferable DNA of the present invention or a multimer thereof is bound to the target molecule, the intracellular transfer region contained in the intracellular transferable DNA is preferably single-stranded. When the intracellular translocation region is annealed with a nucleic acid containing a complementary or substantially complementary base sequence to become double-stranded, the intracellular translocation ability of the intracellular translocation DNA of the present invention is reduced or ineffective. Because it becomes.

The composition of the present invention contains the intracellular migration DNA of the present invention or a multimer thereof as an active ingredient, that is, as a carrier for introducing a target molecule into cells. The composition of the present invention may be composed only of intracellularly-translocating DNA or a multimer thereof, and a target molecule to be introduced into the cell. These solvents, additives or pharmaceutically acceptable carriers may be contained as appropriate. For example, the composition of the present invention may contain a suitable solvent for the intracellularly transferred DNA of the present invention, such as water and a buffer (for example, phosphate buffer, carbonate buffer, Tris buffer) and the like.

The content of the intracellular translocation DNA of the present invention or the multimer thereof contained in the composition of the present invention can be determined by those skilled in the art from the effect of introducing the target molecule into the cell. It can be determined as appropriate.

As used herein, pharmaceutically acceptable carriers include diluents or excipients such as maltose, mannitol, lactose, xylose, trehalose, sorbitol, gelatin, gum arabic, guar gum, tragacanth, ethanol, physiological Examples include saline and Ringer's solution.

The composition of the present invention may contain additives such as stabilizers, buffers, emulsifiers, tonicity agents, preservatives, etc., if necessary, in addition to the above carrier. Examples of the stabilizer include albumin, gelatin, mannitol, sodium EDTA and the like. Examples of the buffer include sodium citrate, citric acid, and sodium phosphate. Examples of the emulsifier include sorbitan fatty acid ester and glycerin fatty acid ester. Examples of the isotonic agent include sodium chloride, potassium chloride, saccharides and the like. Examples of the preservative include benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like.

The composition of the present invention can also contain other drugs as long as the action of the intracellular DNA of the present invention, which is an active ingredient, is not inhibited. For example, in the case of an injection, a predetermined amount of antibiotic may be contained.

Examples of the dosage form of the composition include parenteral dosage forms such as injections, eye drops, creams, nasal drops, ointments, transmucosal agents, plasters and suppositories, liquids, powders, tablets and granules. Oral dosage forms such as, but not limited to, capsules, sublinguals, lozenges and the like.

When a composition is administered to a subject, the specific dosage will depend on the individual subject based on the degree or severity of the disease, general health, age, weight, sex and tolerance to treatment, etc. For example, it is determined by the judgment of a doctor.

The administration of the composition of the present invention may be either systemic administration or local administration (for example, direct administration to the affected area). The route of administration may be either parenteral or oral, for example, intraperitoneal, intravenous, intraarterial, intrahepatic, intravaginal, intramuscular, intramedullary, intrathecal, transdermal, subcutaneous, intradermal , Intranasal, intestinal, intrabronchial, intrapulmonary, or sublingual.

Further, the composition of the present invention is based on a treatment plan determined by a doctor, for example, at regular intervals, for example, 1, 2, 3, 4, 5, 6, 1 week, 2 weeks, The subject can be administered once to several times or several tens of times at intervals of 3 weeks, 1 month, 2 months, 6 months or 1 year.
With the composition of the present invention, a target molecule can be easily introduced into cells.

<Intracellular introduction method of target molecule>
There is also provided a method for introducing a target molecule into cells using the intracellularly transferred DNA of the present invention or a multimer thereof. The method includes the step of contacting the composition of the present invention with a cell. The method of the present invention can be performed in vitro or in vivo.

In the method of the present invention, the use concentration of the composition of the present invention is not particularly limited, and those skilled in the art can achieve the effect of introducing the intracellular molecule of the present invention into the cell. It can be determined as appropriate. In the case of in vitro, the time for contacting the composition with the cells is not particularly limited, and may be appropriately set between 30 minutes and 24 hours, for example, 1 to 2 hours. In the case of in vitro, the cells may be appropriately cultured after contacting the composition with the cells.

According to the method of the present invention, a target molecule can be easily transferred into a cell simply by bringing the composition containing the cell-translocating DNA of the present invention or a multimer thereof and the target molecule to be introduced into the cell into contact with the cell. Can be introduced.

<Agent>
There is also provided an agent for introducing a target molecule into the cell, which contains the intracellularly transferred DNA of the present invention or a multimer thereof. The agent of the present invention can be used for introducing a target molecule into a cell in vitro or in vivo. For example, the agent of the present invention can be used in vitro as an experimental transfection agent. Alternatively, the agent of the present invention can be used in vivo to deliver a target molecule to a living body for medical purposes.

The agent of the present invention contains the intracellular migration DNA of the present invention or a multimer thereof as an active ingredient, that is, as a carrier for introducing a target molecule into cells. The agent of the present invention may consist only of the intracellular translocation DNA of the present invention or a multimer thereof, or other solvents, additives or pharmaceuticals as long as the action of the intracellular translocation DNA of the present invention is not inhibited. An acceptable carrier or the like may be appropriately contained. For example, the agent of the present invention may contain a suitable solvent for the intracellularly transferred DNA of the present invention, such as water and a buffer (for example, phosphate buffer, carbonate buffer, Tris buffer) and the like.

The agent of the present invention may contain a target molecule to be introduced into cells. The target molecule may or may not be bound to the intracellular migration DNA of the present invention contained in the agent or a multimer thereof.

A person skilled in the art can determine the content of the intracellular translocation DNA of the present invention or a multimer thereof contained in the agent of the present invention, and the intracellular translocation DNA of the present invention has an effect of introducing the target molecule into the cell. Thus, it can be determined as appropriate.
With the agent of the present invention, a target molecule can be easily introduced into cells.

<Kit>
There is also provided a kit for introducing a molecule of interest into the cell, which contains the intracellularly transferred DNA of the present invention or a multimer thereof.

The kit of the present invention may further contain a cell culture medium, a cell culture container, and / or instructions describing the protocol used when introducing the target molecule into the cell. Each component contained in the kit of the present invention is individually or in a state where some or all of the components are mixed, placed in a suitable container, and packaged as a whole in one or more. The kit may further include a target molecule to be introduced into the cell. The target molecule may or may not be bound to the intracellular translocation DNA of the present invention or a multimer thereof contained in the kit.

The target molecule can be easily introduced into cells by the kit of the present invention.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(material)
HEK293T cells (human embryonic kidney cells), HeLa cells (human cervical cancer cells), HepG2 cells (human hepatoma cells), HW cells (mouse white fat precursor cells), HB2 cells (mouse brown fat precursor cells), 3T3-L1 Cells (mouse adipose precursor cells) and MCF-7 cells (human breast cancer cells) were used in the examples. These cells were obtained from the National Institute of Physical and Chemical Research Bioresource Center or the National Institute of Biomedical Innovation JCRB Cell Bank. Unless otherwise stated, cells are grown in D-MEM medium containing 10% fetal bovine serum (FBS, CCB) and 1% PSG (penicillin-streptomycin-glutamine) in a 37 ° C / 5% CO ₂ humidified incubator. I let you. FBS added to the cell culture solution of MCF-7 cells was obtained from Sigma.

The DNA used in this example was synthesized by Thermo Fisher Scientific (formerly Life Technologies) or Greiner Japan.

[Example 1]
Screening for intracellular DNA by Cell SELEX method Intracellular DNA was screened from a single-stranded DNA library using Cell SELEX method. The outline of the Cell SELEX method used in this experiment will be described. In this method, only intracellular transferable DNA was selected by repeating a round of selecting and amplifying DNA transferred into cells from a single-stranded DNA library containing random sequences. One round
Step 1: Preparation of cells (and start library DNA in the first round),
Step 2: Incubation with cell DNA library,
Step 3: Decomposition of DNA bound to the cell surface by trypsin and DNase treatment (Step 3 was not performed in the first and second rounds in this example),
Step 4: Extraction of oligo DNA from cells,
Step 5: PCR amplification of the extracted oligo DNA, and Step 6: Purification of the desired single-stranded DNA from the PCR product. In the second and subsequent rounds, steps 1 to 6 were performed using the single-stranded DNA obtained in the preceding round as a DNA library. In the ninth round, the PCR product obtained in step 5 was cloned and sequenced. The specific method was based on Magalhaes, M. et al., Mol. Ther., Vol. 20, No. 3, pp. 616-624, 2012.

In the Cell SELEX method of this example, HEK293T cells were used in the first to third rounds, HeLa cells were used in the fourth to sixth rounds, and 3T3-L1 cells were used in the seventh to ninth rounds. In order to screen for DNA that can be universally introduced into any cell, multiple types of cells were used in this way.
In each round, steps 1 to 3 are different, and steps 4 to 6 are common. The procedure for each round is described in detail below.

(Round 1)
In the first and second rounds, in order to obtain a wide range of candidate DNA from the start library DNA, not only DNA transferred into cells but also DNA bound to the cell surface was used in the next round. Therefore, trypsin and DNase treatment were not performed in the first and second rounds.

(1) As the start library DNA, a single-stranded DNA library containing various DNA sequences of 39 or 40 bases in length and oligo DNA containing 19 or 21 bases in length at both ends was used. Table 1 shows the five types of oligo DNAs used for the preparation of single-stranded DNA libraries.

Each 1.2 μL of 5 types of oligo DNA (100 μM) shown in Table 1 was mixed with 18 μL of serum-containing D-MEM medium in a 1.5 mL tube in a clean bench to prepare a 24 μL start library DNA solution. The solution was stored in the refrigerator until use.

(2) HEK293T cells were seeded at 2 × 10 ⁵ cells per well in a 24-well plate and cultured overnight. The HEK293T cells were detached when the entire medium was exchanged. Therefore, the operation was performed while leaving 100 μL. The medium was removed so that 100 μL of medium remained per well, 376.7 μL of fresh medium was added, and the cells were incubated for 10 minutes.
(3) 3.3 μL of tRNA (40 ng / μL) was added to the medium and incubated at 37 ° C. for 30 minutes.

(4) 20 μL of 24 μL of the start library DNA solution was added to the medium, and incubated at 37 ° C. for 1 hour.
(5) Remove 400 μL of medium and wash the cells twice with 400 μL of sterile Dulbecco's PBS (D-PBS (-)) (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ ) .
(6) 400 μL of sterilized D-PBS (−) was added to the cells, and all remaining solutions were removed.

(7) As described below, extraction of oligo DNA from cells, PCR amplification of the extracted oligo DNA, and purification of the target single-stranded DNA from the PCR product were performed to obtain a single-stranded DNA solution.
(Extraction of oligo DNA from cells)
Oligo DNA was extracted from the cells using TRIzol (Thermo Fisher Scientific). The extraction method was performed according to the extraction method of total RNA. Specifically, 200 μL of TRIzol was dropped onto the cells, pipetting was performed several times, and then all samples were transferred to 1.5 mL tubes. After standing at room temperature for 10 minutes, 40 μL of chloroform was added to the sample, and the mixture was vortexed for 15 seconds. After standing at room temperature for 2 minutes and 30 seconds, the sample was centrifuged at 12000 × g and 4 ° C. for 15 minutes. Only the sample supernatant was collected, transferred to a new 1.5 mL tube, and 100 μL of isopropanol was added to obtain a mixture. After standing at room temperature for 10 minutes, the mixture was centrifuged at 12000 × g, 4 ° C. for 10 minutes. The supernatant was discarded, and 200 μL of 75% ethanol was added to the pellet, followed by centrifugation at 7500 × g and 4 ° C. for 5 minutes. The supernatant was discarded, and the pellet was dried under reduced pressure for 10 minutes. The pellet was dissolved in 10 μL of MilliQ water to obtain an extracted fraction containing oligo DNA extracted from cells.

(PCR amplification of extracted oligo DNA)
The extracted fraction containing the oligo DNA extracted from the cells contained total RNA, and in order to inhibit subsequent PCR amplification, the extracted fraction was subjected to RNase treatment, and then PCR was performed. Specifically, a mixture of 10 × PCR buffer 20 μL, MilliQ water 157 μL, RNase 1 μL, and extracted fraction 2 μL was incubated at 37 ° C. for 30 minutes, and the extracted fraction was subjected to RNase treatment. In the above mixture, dNTP 16 μL, FITC-KS primer (5′-FITC-CTCGAGGTCGACGGTATCG-3 ′: SEQ ID NO: 26) 2 μL, biotin-lambda gt10 primer (5′-Biotin-AGCAAGTTCAGCCTGGTTAAG-3 ′: SEQ ID NO: 27) 2 μL, And Paq5000 DNA Polymerase (Agilent Technology) 1 μL (200 μL scale), PCR amplification was performed by PCR amplification under the following PCR conditions: 25 cycles of 95 ° C. for 20 seconds, 50 ° C. for 20 seconds, and 72 ° C. for 5 seconds. . It was confirmed by 2% agarose gel electrophoresis that the target DNA was amplified.

(Purification of target single-stranded DNA from PCR products)
FITC is bound to the DNA strand having the ability to move into the cell contained in the PCR product (double-stranded DNA), and biotin is bound to the other DNA strand (complementary strand). By fixing double-stranded DNA to streptavidin beads and dissociating the double-stranded DNA into single-stranded DNA with an alkaline solution, only the biotin-bound strand remains on the bead and the other strand remains in solution. Isolate. By this method, the desired single-stranded DNA having the ability to move into cells was purified from the PCR product.

Specifically, first, double-stranded DNA was isolated from the PCR product by magnetic beads. Dynabeads (registered trademark) MyOne ^™ Streptavidin C1 (Thermo Fisher Scientific) was suspended by vortex for 30 seconds, and 40 μL was transferred to a 1.5 mL tube. 1 mL of 1x Binding and Wash buffer (B & W buffer) was added to the tube, and suspended by vortex for 30 seconds. The tube was placed on 16-Position Magnetic Stand (Thermo Fisher Scientific) and allowed to stand for 30 seconds, then the supernatant was discarded and the beads were washed. The beads were washed twice more. To the tube, 2 × B & W buffer 180 μL and PCR product 180 μL were added. The tube was incubated for 15 minutes at room temperature while rotating. A tube was placed on the 16-Position Magnetic Stand for 2 minutes, the supernatant was discarded, and the beads were washed. The beads were further washed 3 times to obtain beads to which double-stranded DNA was bound. Next, 50 μL of 0.2 M NaOH was added to the beads, vortexed and incubated for 1 hour at room temperature. The supernatant was transferred to a new tube to obtain a solution containing the target single-stranded DNA.

Next, the solution containing the target single-stranded DNA was purified by gel filtration using Sephadex® G-50. Specifically, a hole was made with a needle in the bottom of a 1.5 mL tube, and cotton dampened with distilled water was filled in the bottom hole. A new 1.5 mL tube was inserted as a saucer for the perforated tube. 1.5 mL Sephadex® G-50 was placed in a tube with a hole and the tube was centrifuged at 800 × g for 1 minute at room temperature. 300 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) was placed in a tube, and the tube was centrifuged at 800 × g for 1 minute at room temperature to wash Sephadex G-50. Sephadex® G-50 was washed once more. 2 μL of Orange-G was added to the solution containing the single-stranded DNA and slowly dropped onto the Sephadex® G-50 gel. The tube was centrifuged at 800 x g for 1 minute at room temperature. The bottom tube used as the pan was replaced with a new tube, and 50 μL of TE buffer was slowly added dropwise to Sephadex® G-50 gel, and the eluate was collected by centrifugation at 800 × 室温 g for 1 minute at room temperature. The dropping of TE buffer and the collection of the eluate were repeated until Orange-G eluted in the eluate collected in the lower tube. Measure the fluorescence intensity of FITC (excitation wavelength: 488 nm, fluorescence wavelength: 530 nm) of each eluate with a fluorescence plate reader, and mix the eluate with (fluorescence intensity value)> 1 to contain the target single-stranded DNA Got.

Subsequently, the solution containing the target single-stranded DNA was further purified by ethanol precipitation. The pellet obtained by ethanol precipitation according to a standard procedure was dissolved in MilliQ water to a desired concentration to obtain a solution containing purified target single-stranded DNA.

(2nd round)
(1) The first round (2) to (3) was performed in the same manner.
(2) 20 μL of the single-stranded DNA solution obtained in the first round was added to the medium and incubated at 37 ° C. for 1 hour.
(3) The first round (5) to (6) was performed in the same manner.
(4) As described in the first round, extraction of oligo DNA from cells, PCR amplification of the extracted oligo DNA, and purification of the target single-stranded DNA from the PCR product yielded a single-stranded DNA solution. It was.

(3rd round)
From the third round onwards, in order to obtain only the DNA transferred into the cells, the cells were treated with trypsin and DNase after the incubation of the DNA and the cells.
(1) The second round (1) to (3) was performed in the same manner.
(2) To the cells, 170 μL of trypsin was added and incubated at 37 ° C. for 3 minutes.
(3) 750 μL of D-MEM medium was added to the cells, and the solution containing the cells was transferred to a 1.5 mL tube.
(4) The tube was centrifuged at 1000 rpm for 3 minutes at room temperature.

(5) The supernatant was discarded, and 50 μL of D-MEM medium was added to the cells and suspended to obtain a cell suspension.
(6) 0.5 μL of DNase Benzonase (Merck Millipore) solution was added to the cell suspension.
(7) The cell suspension was incubated for 30 minutes at 37 ° C.
(8) The cell suspension was centrifuged at 1000 rpm at room temperature for 3 minutes.
(9) The supernatant was discarded, and 100 μL of sterilized D-PBS (−) was added to the cells, followed by centrifugation at 1000 rpm at room temperature for 3 minutes.
(10) The supernatant was discarded.
(11) As described in the first round, extraction of oligo DNA from cells, PCR amplification of the extracted oligo DNA, and purification of the target single-stranded DNA from the PCR product yielded a single-stranded DNA solution. It was.

(4th and 5th round)
The same operation as in the third round was repeated except that HeLa cells were used instead of HEK293T cells.

(6th round)
(1) HeLa cells were cultured in a 25 cm ² flask for cell culture until they became confluent.
(2) 1 mL of D-PBS (−) containing 5 mM EDTA was added to the flask of (1) and incubated at 37 ° C. for 3 minutes.
(3) 5 mL of D-MEM medium was added to the cells, transferred to a 15 mL tube, and centrifuged at 1000 rpm at room temperature for 3 minutes.
(4) The supernatant was discarded, and 4 mL of D-MEM medium was added to the cells and suspended.
(5) The number of cells was counted with a hemocytometer.

(6) 4 × 10 ⁶ cells were transferred to a 1.5 mL tube.
(7) The tube was centrifuged at 1000 rpm at room temperature for 3 minutes, the supernatant was discarded, and 100 μL of D-MEM medium was added to the cells and suspended to obtain a cell suspension.
(8) 1 μL of tRNA (40 ng / μL) was added to the cell suspension and incubated at 37 ° C. for 10 minutes with inversion mixing.
(9) 20 μL of the single-stranded DNA solution obtained in the previous round was added to the cell suspension and incubated at 37 ° C. for 1 hour.

(10) The cell suspension was centrifuged at 1000 rpm at room temperature for 3 minutes, and the supernatant was discarded.
(11) A mixture of 1 μL Benzonase and 100 μL trypsin was added to the cells, and the cells were incubated at 37 ° C. for 3 minutes.
(12) 500 μL of D-MEM medium was added to the cells, centrifuged at 1000 rpm at room temperature for 3 minutes, and the supernatant was discarded.
(13) The cells were washed twice with 400 μL of sterile D-PBS (−) to remove all of the sterile D-PBS (−).
(14) As described in the first round, extraction of oligo DNA from cells, PCR amplification of the extracted oligo DNA, and purification of the target single-stranded DNA from the PCR product yielded a single-stranded DNA solution. It was.

(Rounds 7 and 8)
The same operation as in the sixth round was repeated except that 3T3-L1 cells were used instead of HeLa cells.

(9th round)
The same operation as in the eighth round was performed until PCR amplification, and the obtained PCR product was cloned into a SmaI-digested pUC18 vector by a standard procedure, and the nucleotide sequence was determined.

(result)
Table 11 shows the 11 base sequences obtained by the Cell SELEX method. Each base sequence was named QAp1, QAp3, QAp4, QAp7, QAp8, QAp10, QAp11, QAp14, QAp15, QAp16 or QAp19. The 11 base sequences were very similar. In addition, 7 of the 19 nucleotide sequences determined were QAp1 sequences.

[Example 2]
Evaluation of DNA intracellular translocation ability By measuring the fluorescence intensity of cells incubated with FITC fluorescently labeled DNA using a flow cytometer, the intracellular translocation ability of the DNA having the base sequence identified in Example 1 was evaluated. did.
(Method)
(1) The oligo DNA shown in Table 3 was commissioned to Greiner Japan. These oligo DNAs (antiKS-QAp1, antiKS-QAp3, antiKS-QAp4, antiKS-QAp7, antiKS-QAp8, antiKS-QAp10, antiKS-QAp11, antiKS-QAp14, antiKS-QAp15, antiKS-QAp16, and antiKS-QAp19) Is an antiKS sequence (shown in italics) on the 5 ′ side, followed by a spacer sequence (shown in lower case), and on the 3 ′ side, each base sequence identified in Example 1 (QAp1, QAp3, QAp4, QAp7 Sequence, QAp8 sequence, QAp10 sequence, QAp11 sequence, QAp14 sequence, QAp15 sequence, QAp16 sequence or QAp19 sequence) (shown in capital letters).

Preparation of monomer: オ リ ゴ FITC-KS primer (5'-FITC-CTCGAGGTCGACGGTATCG-3 ': SEQ ID NO: 26), which is an oligo DNA having FITC at the 5' end, in the oligo DNA (antiKS-QAp1) shown in Table 3 Was annealed to obtain a test DNA monomer (QAp1 monomer). The structure of the QAp1 monomer is shown in FIG. 2A. Specifically, antiKS-QAp1 (SEQ ID NO: 28) (50 μM) 0.8 μL, FITC-KS primer (SEQ ID NO: 26) (50 μM) 0.8 μL, and D-PBS (−) 18.4 μL were mixed at 95 ° C., 80 The test DNA monomer (QAp1 monomer) was prepared by annealing at 5 ° C., 70 ° C., 60 ° C., 55 ° C. and 37 ° C. for 5 minutes each, and stored at 4 ° C.

In addition, in the QAp1 monomer, a nucleic acid whose QAp1 sequence forms a double strand with a complementary oligo DNA antiQAp1 (5'-CGACCCACGCATCCACCGAACCTCCCCCACCACCCCGCCC-3 ': SEQ ID NO: 39) is used as a control DNA monomer. Used as. Specifically, antiKS-QAp1 (SEQ ID NO: 28) (50 μM) 0.8 μL, antiQAp1 (SEQ ID NO: 39) (50 μM) 0.8 μL, FITC-KS primer (SEQ ID NO: 26) (50 μM) 0.8 μL and D-PBS ( -) 17.6 μL of was mixed and annealed at 95 ° C., 80 ° C., 70 ° C., 60 ° C., 55 ° C. and 37 ° C. for 5 minutes each, and a control DNA monomer was prepared and stored at 4 ° C.

Trimer preparation: Each oligo DNA shown in Table 3 (antiKS-QAp1, antiKS-QAp3, antiKS-QAp4, antiKS-QAp7, antiKS-QAp8, antiKS-QAp10, antiKS-QAp11, antiKS-QAp14, antiKS-QAp15 , AntiKS-QAp16, and antiKS-QAp19) are oligo DNA backboneM13Fx3 (5'-TGTAAAACGACGGCCAGTTTGTAAAACGACGGCCAGTTTGTAAAACGACGGCCAGT-3 ': SEQ ID NO: 40), and FITC-KS-antiM13F (5) Annealed with '-FITC-TCGAGGTCGACGGTATCGTACTGGCCGTCGTTTTACA-3': SEQ ID NO: 41) to give test DNA trimers (QAp1 trimer, QAp3 trimer, QAp4 trimer, QAp7 trimer, QAp8 trimer, respectively) , QAp10 trimer, QAp11 trimer, QAp14 trimer, QAp15 trimer, QAp16 trimer or QAp19 trimer). The structure of the test DNA trimer (QAp1 trimer as an example) is shown in FIG. 2B. Specifically, each oligo DNA shown in Table 3 (50 μM) 、 2.4 μL, backboneM13Fx3 (SEQ ID NO: 40) (50 μM) 0.8 μL, FITC-KS-antiM13F (SEQ ID NO: 41) (50 μM) 2.4 μL and D-PBS (-) 14.4 μL of was mixed, annealed at 95 ° C, 80 ° C, 70 ° C, 60 ° C, 55 ° C and 37 ° C for 5 minutes each to prepare a test DNA trimer and stored at 4 ° C .

In addition, in the QAp1 trimer, a nucleic acid in which the QAp1 sequence forms a double strand with antiQAp1 (SEQ ID NO: 39), which is a complementary oligo DNA, was used as a control DNA trimer. Specifically, antiKS-QAp1 (SEQ ID NO: 28) (50 μM) 2.4 μL, antiQAp1 (SEQ ID NO: 39) (50 μM) 2.4 μL, backboneM13Fx3 (SEQ ID NO: 40) (50 μM) 0.8 μL, FITC-KS-antiM13F (sequence) Number 41) (50 μM) 2.4 μL and D-PBS (−) 12 μL were mixed and annealed at 95 ° C., 80 ° C., 70 ° C., 60 ° C., 55 ° C. and 37 ° C. for 5 minutes each, It was taken as a mass.

(2) The medium in the 25 cm ² cell culture flask was removed with an aspirator, and the cells were washed with 1 mL of sterile D-PBS (−).
(3) 1 mL of D-PBS (−) containing 0.5 mM EDTA or trypsin was added and incubated at 37 ° C. until the cells were detached.
(4) 5 mL of D-MEM medium containing 10% FBS and 1% PSG was added, the cells were suspended, and transferred to a 15 mL tube.
(5) The tube was centrifuged at 1000 rpm for 3 minutes at room temperature, and the supernatant was discarded.
(6) The cells were suspended in a serum-containing medium or a serum-free medium so that the desired number of cells was obtained to obtain a cell suspension.

(7) 80 μl of cell suspension was dispensed into a 1.5 mL tube.
(8) The test DNA monomer or trimer prepared in (1) or the control DNA monomer or trimer was added to the cell suspension and incubated at 37 ° C. for 1 hour.
(9) The mixture was centrifuged at 1000 rpm for 3 minutes at room temperature, and the supernatant was discarded.
(10) After adding 100 μL of trypsin and Benzonase 25U to the cells and incubating at 37 ° C. for 3 minutes, 500 μL of D-MEM medium was added to the cells, centrifuged at 1000 rpm for 3 minutes at room temperature, and the supernatant was discarded. However, this step (trypsin and Benzonase (DNase) treatment) was performed only when specified in the “Results” section.
(11) 500 μL of sterilized D-PBS (−) was added to the cells to suspend the cells.

(12) The fluorescence intensity of the cells was measured using a flow cytometer. When the fluorescence intensity value at which the cell count reaches a peak is increased in the test DNA monomer compared to the control DNA monomer, or in the test DNA trimer compared to the control DNA trimer The test DNA monomer or trimer can be regarded as having the ability to translocate into cells. In addition, the ratio of average fluorescence intensity ([average fluorescence intensity of cells incubated with test DNA monomer] / [average fluorescence intensity of cells incubated with control DNA monomer] or [cells incubated with test DNA trimer] Mean fluorescence intensity] / [mean fluorescence intensity of cells incubated with control DNA trimer]). When this ratio exceeds 1, for example, when it is 1.5 or more, the test DNA monomer or trimer can be regarded as having the ability to translocate into the cell.

(Monomer results)
In HepG2 cells incubated with QAp1 monomer, the fluorescence intensity value showing a peak cell count increased compared to the control (FIG. 3A) (average fluorescence intensity ratio 7.2). Also in 3T3-L1 cells incubated with QAp1 monomer, the fluorescence intensity value showing a peak cell count increased compared to the control (FIG. 3B) (average fluorescence intensity ratio 4.6). Therefore, it was shown that the QAp1 monomer can have intracellular translocation ability.

In the experiment whose result is shown in FIG. 3, FITC fluorescence bound to DNA bound to the cell surface may also be detected. Therefore, in order to eliminate the influence of FITC fluorescence bound to DNA bound to the cell surface, the fluorescence intensity was measured after treating the cells with trypsin and DNase. As a result, in 3T3-L1 cells incubated with QAp1 monomer, it was confirmed that the fluorescence intensity value showing the peak cell count still increased compared to the control (FIG. 4) (average fluorescence intensity). Ratio 4.2). Therefore, it was shown that the QAp1 monomer has the ability to move into cells.
From the above results, it was shown that the DNA monomer having the base sequence identified in Example 1 has the ability to migrate into a plurality of cells.

(Trimer results)
QAp1 trimer, QAp3 trimer, QAp4 trimer, QAp7 trimer, QAp8 trimer, QAp10 trimer, QAp11 trimer, QAp14 trimer, QAp15 trimer, QAp16 trimer or In HB2 cells incubated with any of the QAp19 trimers, the fluorescence intensity value showing the peak cell count increased compared to the control (FIG. 5) (ratio of average fluorescence intensity: QAp1 trimer 3.1, QAp3 trimer 1.8, QAp4 trimer 2.0, QAp7 trimer 2.0, QAp8 trimer 3.0, QAp10 trimer 2.9, QAp11 trimer 2.6, QAp14 trimer 1.7, QAp15 trimer 2.6, QAp16 trimer Mer 2.7 and QAp19 trimer 1.8).

When QAp1 trimer was further incubated with HEK293T cells and HW cells, the fluorescence intensity value showing a peak cell count increased in each cell as compared to the control (FIG. 6) (each average Fluorescence intensity ratio 2.5 and 3.8).

From the above, QAp1 trimer, QAp3 trimer, QAp4 trimer, QAp7 trimer, QAp8 trimer, QAp10 trimer, QAp11 trimer, QAp14 trimer, QAp15 trimer, It has been shown that QAp16 trimer and QAp19 trimer can have intracellular translocation ability. It was also shown that the DNA trimer can translocate into HW cells and HB2 cells with low gene transfer efficiency with a commercially available transfection agent.

In the experiments whose results are shown in FIGS. 5 and 6, FITC fluorescence binding to DNA bound to the cell surface may also be detected. Therefore, in order to eliminate the influence of FITC fluorescence bound to DNA bound to the cell surface, the fluorescence intensity was measured after treating the cells with trypsin and DNase. As a result, in HEK293T cells, HB2 cells and HW cells incubated with QAp1 trimer, it was confirmed that the fluorescence intensity value showing the peak cell count still increased compared to the control (FIG. 7) ( Average fluorescence intensity ratios 2.8, 2.1 and 2.6, respectively. Therefore, it was shown that QAp1 trimer has the ability to translocate into cells.
From the above results, it was shown that the DNA trimer having the base sequence identified in Example 1 has the ability to migrate into a plurality of cells.

[Example 3]
Further evaluation of intracellular translocation ability The intracellular translocation ability of DNA containing one or two partial base sequences (5′-side halves) of QAp1 was evaluated.
(Method)
Table 4 shows the oligo DNAs used in this example. These oligo DNAs (antiKS-QAp1-A and antiKS-QAp1-AA) are 5′-side antiKS sequences (shown in italics), followed by spacer sequences (shown in lowercase letters), and 3′-side in Example 1 One or two base sequences (indicated by capital letters) of the 5 ′ half of QAp1 identified in 1.

An oligo DNA shown in Table 4 was annealed with a FITC-KS primer (SEQ ID NO: 26) having FITC at the 5 ′ end to obtain a test DNA monomer (referred to as QAp1A monomer or QAp1AA monomer, respectively) ). Specifically, each oligo DNA shown in Table 4 (50 μM) 0.8 μL, FITC-KS primer (SEQ ID NO: 26) (50 μM) 0.8 μL and D-PBS (−) 18.4 μL were mixed, 95 ° C., 80 The test DNA monomers were prepared by annealing at 5 ° C., 70 ° C., 60 ° C., 55 ° C. and 37 ° C. for 5 minutes each, and stored at 4 ° C.

In addition, FITC-KS primer (SEQ ID NO: 26) was used as a control DNA.

In the same procedure as in (2) to (12) of Example 2, the cells were incubated with the test DNA monomer or the control DNA, and the fluorescence intensity of the cells was measured using a flow cytometer. When the fluorescence intensity value at which the cell count reaches a peak increases in the test DNA monomer as compared to the control DNA, the test DNA monomer can be regarded as having the ability to migrate into the cell. The ratio of average fluorescence intensity ([average fluorescence intensity of cells incubated with test DNA monomer] / [average fluorescence intensity of cells incubated with control DNA]) was calculated. When this ratio exceeds 1, for example, when it is 1.5 or more, the test DNA monomer can be regarded as having the ability to translocate into the cell.

(result)
MCF-7 cells were incubated with each test DNA monomer or control DNA, the cells were treated with trypsin and DNase, and the fluorescence intensity was measured with a flow cytometer. In MCF-7 cells incubated with QAp1A monomer, Compared with the control, the fluorescence intensity value showing a peak in the cell count number hardly increased (FIG. 8A) (average fluorescence intensity ratio 1.2). Therefore, it was shown that a DNA monomer containing only one 5'-side base sequence of QAp1 does not have the ability to move into cells. On the other hand, in the MCF-7 cells incubated with the QAp1AA monomer, the fluorescence intensity value showing the peak cell count increased compared to the control (FIG. 8B) (average fluorescence intensity ratio 2.4). Therefore, it was shown that DNA containing two base sequences of the 5 ′ half of QAp1 has the ability to move into cells.

From the above, it was shown that DNA containing two 5′-side base sequences of the base sequence identified in Example 1 has the ability to migrate into cells.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (9)

1. The following base sequence:
   5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-N22-N23-TCG-N27-N28-N29-G-N31-AT-N34-N35-GTG -N39-N40-TCG-3 '
   [here,
   N1 is deleted or T,
   N2 is G or is deleted;
   N5 is C or T;
   N6 is G or C;
   N7 is G or T,
   N14 is G or C;
   N19 is A or G;
   N20 is G or A,
   N22 is deleted or T,
   N23 is T or A,
   N27 is G, T or A,
   N28 is T, A or C;
   N29 is deleted or T,
   N31 is G or A;
   N34 is G or is deleted;
   N35 is C or A,
   N39 is G or T, and N40 is G or A] (SEQ ID NO: 1)
   Intracellular DNA comprising an intracellular translocation region consisting of
2. The intracellular transferable DNA according to claim 1, wherein the base sequence is the following (a) or (b).
   (a) 5'-GGGCGGGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 2),
   5'-GGTGGGGTGGTGGGGGAGGTTCGTTGGATGCGTGGGTCG-3 '(SEQ ID NO: 3),
   5'-GGGCGGGGTGGTCGGGGGAGTTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 4),
   5'-GGGCGGGGTGGTGGGGGAGGTTCGTATGGATGCGTGGATCG-3 '(SEQ ID NO: 5),
   5'-GGGCGGGGTGGTGGGGGAGGTTCGATGGATCGTGGGTCG-3 '(SEQ ID NO: 6),
   5'-GGGCGGGGTGGTGGGGGAGGATCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 7),
   5'-GGCGGGGTGGTGGGGGAGGTTCGATGGATGAGTGGGTCG-3 '(SEQ ID NO: 8),
   5'-GGGCCGGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3 '(SEQ ID NO: 9),
   5'-GGGCGGGGTGGTGGGGGAGGTTCGACGGATGCGTGGGTCG-3 '(SEQ ID NO: 10),
   5'-TGGGCGGGGTGGTGGGGGAGGTTCGGTGAATGCGTGTGTCG-3 '(SEQ ID NO: 11), and 5'-GGGCGTGGTGGTGGGGGAGGTTCGGTGGATGCGTGGGTCG-3' (SEQ ID NO: 12)
   (B) a base sequence in which one or two bases are substituted, deleted, inserted or added in the base sequence shown in any of SEQ ID NOs: 2 to 12
3. The following base sequence:
   5'-N1-N2-GG-N5-N6-N7-GGTGGT-N14-GGGG-N19-N20-G-3 '
   [here,
   N1 is deleted or T,
   N2 is G or is deleted;
   N5 is C or T;
   N6 is G or C;
   N7 is G or T,
   N14 is G or C;
   N19 is A or G, and N20 is G or A] (SEQ ID NO: 13)
   Intracellular DNA comprising an intracellular translocation region containing 2 or more of.
4. The intracellular DNA according to claim 3, wherein each of the base sequences is the following (a) or (b).
   (a) 5'-GGGCGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 14),
   5'-GGTGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 15),
   5'-GGGCGGGGTGGTCGGGGGAG-3 '(SEQ ID NO: 16),
   5'-GGCGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 17),
   5'-GGGCCGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 18),
   5'-TGGGCGGGGTGGTGGGGGAGG-3 '(SEQ ID NO: 19), and 5'-GGGCGTGGTGGTGGGGGAGG-3' (SEQ ID NO: 20)
   (B) a base sequence in which one base is substituted, deleted, inserted or added in the base sequence shown in any of SEQ ID NOS: 14 to 20
5. A multimer of intracellular translocating DNA formed by binding two or more intracellular translocating DNAs, each of the intracellular translocating DNAs according to any one of claims 1 to 4. Intracellular DNA multimer selected from intracellular translocation DNA.
6. 5. An agent for introducing a target molecule into the cell, comprising the intracellular transferable DNA according to any one of claims 1 to 4 or the intracellular transferable DNA multimer according to claim 5.
7. 5. A composition comprising the intracellular transferable DNA according to any one of claims 1 to 4 or the intracellular transferable DNA multimer according to claim 5 and a target molecule to be introduced into the cell.
8. A method for introducing a target molecule into a cell, comprising contacting the composition according to claim 7 with the cell.
9. A kit for introducing a target molecule into the cell, comprising the intracellular transferable DNA according to any one of claims 1 to 4 or the intracellular transferable DNA multimer according to claim 5.

Images