CN103180440B - Method and the nucleic acid complexes of nucleic acid is imported in cell - Google Patents
Method and the nucleic acid complexes of nucleic acid is imported in cell Download PDFInfo
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Abstract
The invention discloses a kind of method importing nucleic acid in cell and the nucleic acid complexes for it, described method possesses following operation: have basic amino acid and the alternately arranged aminoacid sequence of hydrophobic amino acid, and have in the presence of double-stranded double chain acid molecule, the nucleic acid carrier with following peptide chain is made to contact with double chain acid molecule, via the side chain of basic amino acid and the electrostatic interaction of phosphate and the one or both of hydrogen bond, aforementioned double chain acid molecule and aforementioned peptide chain combination is made to form nucleic acid complexes, wherein, the side chain of the basic amino acid that described peptide chain formation has positive charge is arranged in one side side, the side chain of hydrophobic amino acid is arranged in the β-pleated sheet structure of side, opposite.
Description
Technical field
The present invention relates to import the method for double chain acid molecule in target cell, for the changing of nucleic acid carrier of the method
Nucleic acid complexes that is good and that utilize aforementioned nucleic acid carrier and double chain acid molecule to obtain.
Background technology
RNA interference (RNAi) be the low-molecular-weight double-stranded RNA (siRNA) with base a length of about 21 ~ 25 as inducement, base
Sequence-specific ground decomposes mRNA, thus suppresses the phenomenon of the expression of specific gene, and comes into the anti-of clinical experimental stage
Phosphorothioate odn is compared, and effectiveness and the base sequence specificity of RNA interference are more excellent, the most strongly expects its answering in medicine
With.But, also remain siRNA to intracellular importing, at the guaranteeing of persistence of intracellular stability and inhibition
Deng a large amount of problems needing to solve.
It is polyanion owing to comprising poly-(few) nucleotide of siRNA, when the most directly using, with hydrophobic cell
The affinity of film is low, it is difficult to import intracellular.Further, since nuclease can decompose ectogenic nucleic acid, in order to ensure it carefully
The stability of intracellular and the persistence of inhibition, need to protect it from the impact of the nuclease of intracellular existence.In view of upper
State problem, for nucleic acid to intracellular importing, propose various method up to now, but these methods are roughly divided into use disease
The method of poisonous carrier and the method not using viral vector.
Viral vector refers to be short of the virus of replication capacity, in the method for gene introduction employing viral vector, has
The advantage of Reproduction methods of virus can be utilized, make from enter intracellular advance the most efficiently to protein synthesis such excellent
Point.As viral vector, use derives from adenovirus, retrovirus retrovirus, slow virus, (ア デ ノ is with companion ウ イ for adeno-associated virus
Le ス) recombinant viral vector (for example, referring to patent documentation 1, non-patent literature 1).But, use the method quilt of viral vector
Point out to exist because of virus the danger of carcinogenic grade, the most also confirm to there is death.Additionally, there are because producing in virus
The problems such as inactivation are caused, the problem being simultaneously accompanied by being difficult to produce, be difficult to qualitative control in a large number with antibody.
On the other hand, as the method not using viral vector and import nucleic acid in target cell, it is proposed that calcium phosphate method,
Electroporation, liposome method, fat transfect thing method, microinjection, hydrodynamic, utilize the carrier such as antibody, peptide and nucleic acid
The various methods (for example, referring to non-patent literature 1) such as the method for complex.
But, do not use in the existing nucleic acid delivery methods of viral vector, thin for the nucleic acid headed by siRNA is imported
Gene medicine in born of the same parents builds cannot say it is sufficient.I.e., seek not limit cell category, have realization concurrently efficiently and with Gao Chong
Existing property imports nucleic acid, improves the patience to intracellular nuclease, is combined and affine with target nucleic acid with improving sequence-specific
The novel nucleic acid vectors of the character such as property, low cytotoxicity.
Cut-off so far, develops the various method importing nucleic acid in nucleic acid carrier, cell, in recent years, employs biology
The functional peptides such as internal signal peptide, the method that imports nucleic acid in nucleic acid carrier and cell gets most of the attention.Such as have following
Method: use nuclear location signal peptide (for example, referring to patent documentation 2,3), other signal peptide (for example, referring to patent documentation 4),
Designed peptide (for example, referring to patent documentation 5), to the method for intracellular importing anionic property nucleic acid；Be applied in combination utilize sugar-modified
Peptide makes the merit that nucleic acid carrier (for example, referring to patent documentation 6) that cell-specific improves, amphipathic nature polyalcohol are combined into peptide
The method (for example, referring to patent documentation 8) etc. of energy property molecule (for example, referring to patent documentation 7), existing nucleic acid carrier and peptide.
Prior art literature
Patent documentation
Patent documentation 1: Japanese Unexamined Patent Publication 10-146191 publication
Patent documentation 2: Japanese Unexamined Patent Application Publication 2003-514564 publication
Patent documentation 3: Japanese Unexamined Patent Publication 2001-288200 publication
Patent documentation 4: Japanese Kohyo 10-506001 publication
Patent documentation 5: Japanese Unexamined Patent Publication 2002-316997 publication
Patent documentation 6: Japanese Unexamined Patent Publication 11-290073 publication
Patent documentation 7: Japanese Unexamined Patent Application Publication 2003-503370 publication
Patent documentation 8: Japanese Unexamined Patent Publication 2004-65238 publication
Non-patent literature
Non-patent literature 1: secondary island one model, Bei Cunyi be great to be write, " must enter と and resolve プ by ず left-hand seat く い く
ロ ト U Le ", Yang Tushe, in JIUYUE, 2003, ISBN9784897064116
Summary of the invention
As it has been described above, cut-off is so far, develops the method importing nucleic acid in various nucleic acid carriers and cell, but there is no
The method being sized.It addition, mostly with DNA as target in these methods, be therefore not necessarily suitable for low-molecular-weight and reactivity
The siRNA higher than DNA is directed in cell.
The present invention is carried out in view of described situation, its object is to, it is provided that a kind of side importing nucleic acid in cell
Method, nucleic acid carrier and nucleic acid complexes, the double chain acid molecules such as siRNA can be imported in target cell by described method efficiently,
The high patience to nuclease can be given to double chain acid molecule, and the side effect risk brought by cytotoxicity is little.
For realize first embodiment of the present invention of object defined above by provide described in following (1) ~ (9) to cell
The method of middle importing nucleic acid solves the problems referred to above.
(1) a kind of method importing nucleic acid in cell, the method possesses following operation: has basic amino acid and is dredging
Aminoacid sequence that aqueous aminoacid is alternately arranged also has in the presence of double-stranded double chain acid molecule, makes
Have to be formed and there is the amino acid whose side chain of aforementioned base of positive charge be arranged in one side side, the side chain of aforementioned hydrophobic amino acid
The nucleic acid carrier of the peptide chain being arranged in the beta sheet structure of side, opposite contacts with aforementioned double chain acid molecule, via aforementioned base
Hydrogen bond between amino acid whose side chain and the electrostatic interaction of phosphate and aforementioned double chain acid molecule and aforementioned peptide chain
One or both, makes aforementioned double chain acid molecule and aforementioned peptide chain combination form the operation of nucleic acid complexes, and, by aforementioned
Nucleic acid complexes imports the operation of cell.
(2) according to the method importing nucleic acid in cell described in (1), wherein, aforementioned base aminoacid choosing freely essence ammonia
Acid, lysine and the group of ornithine composition, aforementioned hydrophobic amino acid selects free leucine, isoleucine, valine and color ammonia
The group of acid composition.
(3) according to the method importing nucleic acid in cell described in (1) or (2), wherein, aforementioned peptide chain has (RL)nWith
(LR)nIn any one shown aminoacid sequence.
(wherein, R, L represent arginine residues, leucine residue respectively, and n represents the natural number of more than 5 and less than 10.)
(4) according to the method importing nucleic acid in cell according to any one of (1) ~ (3), wherein, it is being previously formed core
In the operation of acid complex, aforementioned double chain acid molecule is 1:4 ~ 1:30 with the mol ratio of nucleic acid carrier.
(5) according to the method importing nucleic acid in cell according to any one of (1) ~ (4), wherein, aforementioned double-strandednucleic acid
Molecule is siRNA.
(6) according to the method importing nucleic acid in cell according to any one of (1) ~ (5), wherein, aforementioned nucleic acid is combined
Thing has the activity of the localization to target cell and/or its specific part.
(7) according to the method importing nucleic acid in cell described in (6), wherein, aforementioned contained by aforementioned nucleic acid complex
Double chain acid molecule or aforementioned nucleic acid carrier be combined with the location having target cell and/or its specific part at least partially
Change the signal peptide of activity.
(8) according to the method importing nucleic acid in cell described in (7), wherein, aforementioned signal peptide has following sequence
Any one shown aminoacid sequence in number 1 ~ 13,
QAKKKKLDK [serial number 1]
SPQPKKKP [serial number 2]
RQARRNRRRRWR [serial number 3]
GPKKKRKV [serial number 4]
NSAAFEDLRVLS [serial number 5]
RQIKIWFQNRRMKWKKEN [serial number 6]
GRKKRRQRRRPPQG [serial number 7]
LPPLERLTL [serial number 8]
ALQKKLEELELDE [serial number 9]
LALKLAGLDI [serial number 10]
SLEGAVSEISLRD [serial number 11]
LPVLENLTL [serial number 12]
LASLMNLGMS [serial number 13].
(9) according to the method importing nucleic acid in cell described in (7) or (8), wherein, the end of aforementioned signal peptide combines
In the peptide chain contained by aforementioned nucleic acid carrier or the end of aforementioned double-strandednucleic acid.
Second embodiment of the present invention solves above-mentioned by providing the nucleic acid complexes described in following (10) ~ (16)
Problem.
(10) a kind of nucleic acid complexes, wherein, this nucleic acid complexes is for have basic amino acid and hydrophobic amino acid
Alternately arranged aminoacid sequence also has in the presence of double-stranded double chain acid molecule, makes have formation tool
The amino acid whose side chain of aforementioned base having positive charge is arranged in one side side, the side chain of aforementioned hydrophobic amino acid is arranged in opposite
The nucleic acid carrier of the peptide chain of the beta sheet structure of side and double chain acid molecule are via the alkali contained by the peptide chain of aforementioned nucleic acid carrier
The electrostatic interaction of the phosphate of the side chain of acidic amino acid and aforementioned double chain acid molecule and combining obtains.
(11) according to nucleic acid complexes described in (10), wherein, aforementioned base aminoacid select free arginine, lysine and
The group of ornithine composition, aforementioned hydrophobic amino acid selects free leucine, isoleucine, valine and the group of tryptophan composition.
(12) according to the nucleic acid complexes described in (10) or (11), wherein, aforementioned peptide chain has (RL)n(LR)nIn arbitrary
Aminoacid sequence shown in person.
(wherein, R, L represent arginine residues, leucine residue respectively, and n represents the natural number of more than 5 and less than 20.)
(13) according to the nucleic acid complexes according to any one of (10) ~ (12), it is characterised in that aforementioned double chain acid molecule
For siRNA.
(14) according to the nucleic acid complexes according to any one of (10) ~ (13), wherein, aforementioned double chain acid molecule or combination
Being combined with at least partially of aforementioned nucleic acid carrier in it has the activity of the localization to target cell and/or its specific part
Signal peptide.
(15) according to the nucleic acid complexes described in (14), wherein, aforementioned signal peptide has above-mentioned serial number 1 ~ serial number
The aminoacid sequence that in 13, any one is shown.
(16) according to the nucleic acid complexes described in (14) or (15), wherein, the end of aforementioned signal peptide is incorporated into aforementioned core
Peptide chain contained by acid vectors or the end of aforementioned double-strandednucleic acid.
The nucleic acid carrier of the present invention has in the presence of double-stranded double chain acid molecule, has formation and just has
The side chain of the basic amino acid of electric charge is arranged in one side side, the side chain of hydrophobic amino acid is arranged in the beta sheet of side, opposite
The peptide chain of structure.Having report to claim, the double chain acid molecule such as peptide and double-stranded DNA forming beta sheet structure can divide via double-strandednucleic acid
Hydrogen bond etc. between son and peptide and combine, thus formed complex (for example, referring to Proc.Natl.Acad.Sci.USA,
vol.74(1977),p.1458-1462).In peptide chain contained by the nucleic acid carrier of the present invention, due to basic amino acid and hydrophobicity
Aminoacid is alternately arranged, therefore when forming beta sheet structure, have the side chain of basic amino acid of positive charge with about 7nm between
Every the one side side being arranged in beta sheet structure.The interval between phosphate in this interval and nucleotide chain is basically identical.Cause
This, the nucleic acid carrier of the present invention is being formed with at the peptide chain part of beta sheet structure side chain and phosphate via basic amino acid
Electrostatic interaction and double chain acid molecule and aforementioned peptide chain between one or both and double-stranded RNA, the double-strand of hydrogen bond
The double chain acid molecules such as DNA are securely joined with, such that it is able to the nuclease patience of these double chain acid molecules is greatly improved, with
Time, owing to the side chain of hydrophobic amino acid exposes the outside to complex, the cell membrane therefore formed by bimolecular lipid membrane
Permeability improves, such that it is able to improve the efficiency importing double chain acid molecule in cell.It addition, by suitably selecting peptide chain portion
The aminoacid sequence divided, it is also possible to increase and the affinity of double chain acid molecule of nucleic acid carrier, base sequence specificity.Separately
Outward, the nucleic acid carrier of the present invention, owing to polypeptide as substrate, therefore cytotoxicity is low, can reduce and be applied to gene therapy, base
Because of side effect during preparation.And then, by with signal peptide modification etc., it is also possible to realize intracellular localization control, increase thin
Born of the same parents' specificity, can give increase nucleic acid and import the various functions such as the side effect in efficiency, reduction gene therapy.
In the method importing nucleic acid in cell of the present invention, owing to using the nucleic acid carrier with feature as described above
In target cell, import double chain acid molecule, therefore have the effect that and the double chain acid molecules such as siRNA can be led efficiently
Enter in target cell, it is possible to double chain acid molecule is given high patience to nuclease, and the side effect brought by cytotoxicity
Risk is little.
It addition, the nucleic acid complexes of the present invention can import the double chain acid molecules such as siRNA efficiently in target cell
While, also there is the high patience to nuclease.And then, owing to polypeptide as substrate, therefore cytotoxicity is low, can reduce should
Side effect when gene therapy, gene formulations, can be suitably employed in these purposes.
Accompanying drawing explanation
Fig. 1 is siRNA, nucleic acid carrier (RL)7Circular dichroism (CD) spectrogram with nucleic acid complexes.
The efficiency importing nucleic acid complexes in Jurkat cell is produced by Fig. 2 by the peptide chain length in show nucleic acid carrier
The fluorescent microscopy images of raw effect.
Fig. 3 is that the show nucleic acid carrier mol ratio with double chain acid molecule is to importing nucleic acid complexes in Jurkat cell
Efficiency produced by the fluorescent microscopy images of effect and fluorogram.
Fig. 4 is for showing nuclear location signal (NLS) peptide and long-chain carboxylic acid and (RL)7Conjugate pair lead in Jurkat cell
Enter the fluorescent microscopy images of effect produced by the efficiency of nucleic acid complexes.
Fig. 5 is that the show nucleic acid carrier mol ratio with double chain acid molecule is to importing nucleic acid complexes in HeLa cell
The fluorescent microscopy images of effect produced by efficiency and fluorogram.
Fig. 6 is display nuclear export signal (NES) peptide and (RL)7Conjugate pair in HeLa cell, import nucleic acid complexes
The fluorescent microscopy images of effect produced by efficiency.
Fig. 7 is the gel electricity of the rheological parameters' change with time in 10% hyclone (FBS) of the siRNA contained by show nucleic acid complex
The result of swimming.
Fig. 8 is the gel electricity of the rheological parameters' change with time in 10% hyclone (FBS) of the siRNA contained by show nucleic acid complex
The result of swimming.
Fig. 9 is to show survival rate when cultivating Jurkat cell in the presence of nucleic acid carrier and RNAiFect
The figure of rheological parameters' change with time.
Figure 10 is to show survival rate when cultivating K562 cell in the presence of nucleic acid carrier and RNAiFect
The figure of rheological parameters' change with time.
Detailed description of the invention
Then, illustrate for by the embodiment that the present invention embodies, with for understanding the present invention.
The method that what first embodiment of the present invention related to import nucleic acid in cell is (following, sometimes referred to simply as " to carefully
The method importing nucleic acid in born of the same parents " or " this method ".) possess following operation: there is basic amino acid and hydrophobic amino acid friendship
Replace the aminoacid sequence of arrangement and have in the presence of double-stranded double chain acid molecule, making that there is formation and have
The side chain of the basic amino acid of positive charge is arranged in one side side, the side chain of hydrophobic amino acid is arranged in the β-folding of side, opposite
The nucleic acid carrier of the peptide chain of stack structure contacts with double chain acid molecule, via the electrostatic phase of side chain and the phosphate of basic amino acid
The one or both of the hydrogen bond between interaction and double chain acid molecule and peptide chain, makes double chain acid molecule chain with aforementioned peptide
Close and form the operation of nucleic acid complexes, and nucleic acid complexes is imported the operation of cell.
First, (following, sometimes referred to simply as " nucleic acid carrier " for the nucleic acid carrier used in this method.) and the present invention
Nucleic acid complexes that second embodiment relates to (following, sometimes referred to simply as " nucleic acid complexes " or " complex ".) carry out more detailed
Thin explanation.
(1) nucleic acid carrier
There is basic amino acid and the alternately arranged aminoacid sequence of hydrophobic amino acid and there is Double helix knot
In the presence of the double chain acid molecule of structure, nucleic acid carrier has the peptide chain forming beta sheet structure, in described beta sheet structure, and tool
The side chain having the basic amino acid of positive charge is arranged in one side side, the side chain of hydrophobic amino acid is arranged in side, opposite.As
Upper described, the polypeptide chain in beta sheet structure can form complex, especially with the double chain acid molecule such as double-stranded DNA (dsDNA)
It is, if the side chain that peptide chain presents the basic amino acid with positive charge is arranged in the one side side of foldable structure, hydrophobicity ammonia
The side chain of base acid is arranged in the secondary structure of side, opposite, then can be via the electrostatic phase of the side chain of basic amino acid with phosphate
The one or both of the hydrogen bond between interaction and double chain acid molecule and aforementioned peptide chain, forms stable knot between
Close.In the nucleic acid complexes being thusly-formed, the side chain of hydrophobic amino acid, towards outside, therefore divides with by the lipid of complex is double
The affinity of the cell membrane that sub-film is formed increases, and imports intracellular efficiency and improves.
For the aminoacid sequence of peptide chain, as long as meet above-mentioned condition, i.e. meet have basic amino acid and
Aminoacid sequence that hydrophobic amino acid is alternately arranged also has in the presence of double-stranded double chain acid molecule,
It is right that the side chain that can form the basic amino acid with positive charge is arranged in one side side, the side chain of hydrophobic amino acid is arranged in
The beta sheet structure of side, face, via the side chain of basic amino acid and the electrostatic interaction of phosphate and double chain acid molecule
And the one or both of the hydrogen bond between aforementioned peptide chain, forms the condition of stable combination between, limits the most especially
Fixed.As long as it addition, beta sheet structure meets above-mentioned condition, it is also possible to be appointing in parallel beta sheet and anti-parallel ss-sheet structure
A kind of.
The formation of beta sheet structure can use the arbitrary known methods such as circular dichroism (CD) spectrogram to confirm.
As the preferred amino acid sequence of peptide chain, can list and select free arginine, lysine and the group of ornithine composition
In basic amino acid and the hydrophobic amino acid in the group selecting free leucine, isoleucine, valine and tryptophan to form
Alternately arranged aminoacid sequence.The kind of basic amino acid and hydrophobic amino acid can form complex according to being used for
The kind of double chain acid molecule, structure etc. suitably selects.It addition, polypeptide chain length can form complex according to being used for
The chain length of double chain acid molecule, the quantity etc. of phosphate (negative charge) that i.e. double chain acid molecule has suitably selects.
When polypeptide chain is too short relative to double chain acid molecule, it is impossible to form stable complex, protection double-strandednucleic acid cannot be obtained simultaneously
The effect that molecule is not affected by nuclease.On the other hand, when polypeptide chain is long relative to double chain acid molecule, due to basic amine group
The positive charge that acid residue is had, the efficiency importing nucleic acid complexes in cell reduces.
As the aminoacid sequence of preferred peptide chain, (RL) can be listedn(LR)nAminoacid shown in any one of
Sequence.Wherein, R, L represent arginine residues, leucine residue respectively.Arginine (R) residue contains guanidine radicals as basic group,
As shown in chemical formula described as follows, owing to it can interact consumingly with phosphate, therefore forming stable being combined
The aspect of thing is preferred.
[chemical formula 1]
It addition, the natural number that n is the number of repetition representing RL or LR, can be according to the double-strandednucleic acid for forming complex
The quantity etc. of the phosphate (negative charge) that the chain length of molecule, i.e. double chain acid molecule are had suitably selects.Double-strandednucleic acid
During for siRNA, n is such as more than 5, is preferably more than 5 and less than 20, is more preferably the natural number of more than 7 and less than 10.N is low
In 5 time, it is difficult to form stable combination with double chain acid molecule, on the other hand, when n becomes excessive, may produce and manifest carefully
The problem such as cellular toxicity or the negative charge being difficult to complete neutralising phosphoric acid base.
Nucleic acid carrier is preferably to the specific part in target cell and target cell or the one of organelle (intracellular organella)
Person or both have localization activity.About in the target cell of localization object or the target cell that become nucleic acid complexes
Specific part or organelle, can be according to the kind of the double chain acid molecule in target cell to be imported and this intracellular function etc.
Suitably select.
The means giving localization activity can be according to the target cell of localization object or the target becoming nucleic acid complexes
Intracellular specific part or organelle use arbitrary known approaches.Such as, as giving the location to target cell
Change the object lesson of the means of activity, the part corresponding to the receptor being specifically present in this target cell of sening as an envoy to and core can be enumerated
Acid vectors combines.It addition, as the means for giving the activity of the localization to the specific part in target cell or organelle
Object lesson, can enumerate sends as an envoy to has the active signal peptide of the localization to target cell and/or its specific part and nucleic acid carrier
In conjunction with.As the object lesson of combinable signal peptide, various nuclear location signal (NLS) and nuclear export signal can be listed
(NES) etc..
Be achieved by nuclear localization: NLS with as the input albumen of transport protein with as gtp binding protein
One of Ran combine, make this coalition by nuclear Pore Complex identification, thus lead to from Cytoplasm and realize in core.
As the object lesson of NLS, can list and there is any one shown aminoacid sequence in following serial number 1 ~ 7
Peptide.
QAKKKKLDK [serial number 1] (derives from nucleoplasmin (ヌ Network レ オ プ ラ ス ミ Application, nucleoplasmin)
NLS sequence)
SPQPKKKP [serial number 2] (derives from the NLS sequence of human P 53)
RQARRNRRRRWR [serial number 3] (derives from the NLS sequence of HIV-1Rev albumen)
GPKKKRKV [serial number 4] (derives from the NLS sequence of SV40T antigen)
NSAAFEDLRVLS [serial number 5] (derives from the NLS sequence of influenza virus nucleoplasmin)
RQIKIWFQNRRMKWKKEN [serial number 6] (derives from feeler foot cell-penetrating peptide (ア Application テ Na ペ デ ィ ア ペ ネ ト ラ
チ Application, antennapedia penetratin) NLS sequence)
GRKKRRQRRRPPQG [serial number 7] (derives from the NLS sequence of HIV-1Tat albumen)
And be combined as GTP to the cytoplasmic implemented below that is transported through: NES and the CRM1 as transport protein by core
The Ran of one of albumen combines, and makes this coalition by nuclear Pore Complex identification, thus leads to from core and realize into Cytoplasm.
As the object lesson of NES, can list and there is the aminoacid sequence that in following serial number 8 ~ 13, any one is shown
The peptide of row.
LPPLERLTL [serial number 8] (derives from the NES sequence of HIV-1Rev albumen)
ALQKKLEELELDE [serial number 9] (derives from the NES sequence of MAPKK)
LALKLAGLDI [serial number 10] (derives from the NES sequence of PKI-α)
SLEGAVSEISLRD [serial number 11] (derives from the NES sequence of Dsk-1)
LPVLENLTL [serial number 12] (derives from the NES sequence of TFIIIA)
LASLMNLGMS [serial number 13] (derives from the NES sequence of Matrin3)
About these signal peptides, the formation of complex will not suppressed and under conditions of intracellular importing, Ke Yijie
Together in the intramolecular optional position of nucleic acid carrier, such as with N-end side or the C-end of the polypeptide chain of double chain acid molecule bonding
Side, or under conditions of the expression that will not suppress beta sheet structure, side chain can be incorporated into, the N-end side of signal peptide and C-
The either side of end side is combined with double chain acid molecule, it is preferred that the C-end of signal peptide is incorporated into and double-strandednucleic acid
The N-end of the peptide chain of molecule bonding.Can be directly in conjunction with the polypeptide chain of double chain acid molecule bonding and signal peptide, it is also possible to
It is combined via suitable spacer molecule (spacer molecule).As the object lesson of spacer molecule, Asia can be listed
Alkyl, polyoxyethylene, oligopeptide etc..
Nucleic acid carrier can also be in conjunction with functional point in addition to the part corresponding to the receptor on target cell, signal peptide
Son.As the object lesson of functional molecular, can list and for raising, the chain alkyl of affinity, the lipid of cell membrane be divided
Son etc..
Owing to nucleic acid carrier is with polypeptide for main composition key element, therefore cytotoxicity is more existing than cation system liposome etc.
Nucleic acid carrier is low, can reduce side effect when being applied to gene therapy, gene formulations, can be applied suitably to these and use
On the way.
The cytotoxicity of nucleic acid carrier can be by being conducted into target cell the cell survival rate after the stipulated time
Evaluate.
(2) nucleic acid complexes
Electrostatic phase by side chain and the phosphate in double chain acid molecule via the basic amino acid in nucleic acid carrier
Interaction and make both combine, nucleic acid complexes can be obtained.
As the double chain acid molecule for forming complex, can be any one in DNA, RNA, about to intracellular
The purpose imported, for example, it may be used for producing desired protein, use " bait " short-chain nucleic acids catches identification double-stranded DNA
The arbitrarily purpose such as the transcription factor of sequence etc. (bait method, decoymethod) or RNA interference method (RNAi).Accordingly, as double
Chain nucleic acid molecules, can list coding and make it at the double-stranded DNA of the protein of cell inner expression, decoy nucleic acid
(decoynucleic acids), siRNA etc..The base length of double chain acid molecule and base sequence are according to importing core in cell
The acid purpose of complex, target cell, become the kinds of Diseases etc. for the treatment of target and different, in the case of siRNA, base number is 9 ~
50 bases, preferably 15 ~ 30 bases, more preferably 18 ~ 28 bases.In the case of human, if base number is more than 17, then institute
Obtain the sum (4 of polynucleotide17=1.7×1010) exceed the gene number (6 × 10 of people9), so that only suppressing specific gene
Express and be possibly realized statistically.
About base sequence, known at the base sequence of the aminoacid sequence or target gene that become the protein expressing object
In the case of, can use based on the sequence data that can obtain from the data bases such as GenBank, EMBL, PDB, DDBJ
Arbitrary known method, the double chain acid molecule that design imports in target cell.Or, it is also possible to by arbitrary known method
The aminoacid sequence determining the target protein utilizing arbitrary known method to separate or the mRNA's encoding this aminoacid sequence
Base sequence, and design double chain acid molecule according to these sequences.
There is the mechanism of the expression of the target nucleic acid of the base sequence complementary with double chain acid molecule to be imported as suppression,
Can list: (1) is decomposed mRNA/ gather by specifically being hydrolyzed the ribonuclease H of RNA chain in RNA/DNA hybridization chain
Nucleotide complex；(2) translation is suppressed by ribosome complex；(3) polynucleotide connecing with intron and exon
When portion of boundary is target, the montage of suppression mRNA；(4) disturbed by RNA and specifically hydrolyze the base sequence of mRNA；(5) pass through
Decoy nucleic acid suppresses transcription control factors etc..
Nucleic acid complexes is preferably to the specific part in target cell and target cell or organelle (intracellular organella)
One or both has localization activity.About the spy in the localization object i.e. target cell of nucleic acid complexes or target cell
Determine position or organelle, can enter according to the kind of the double chain acid molecule in target cell to be imported and this intracellular function etc.
Row suitably selects.
The means giving localization activity can be according to the localization object i.e. target cell of nucleic acid complexes or target cell
Interior specific part or organelle use arbitrary known approaches.Such as, as alive for giving the localization to target cell
The object lesson of the means of property, is specifically present in joining corresponding to the receptor of this target cell as it has been described above, can enumerate to send as an envoy to
Body is combined with nucleic acid carrier, or makes this part be combined with double chain acid molecule.It addition, as giving in target cell
The object lesson of the means of the localization activity of specific part or organelle, as it has been described above, send as an envoy to signal peptide and nucleic acid can be enumerated
At least some of combination of carrier, or make this signal peptide be combined with double chain acid molecule.It addition, import in nucleic acid complexes
The amount of signal peptide too much time, it is possible to cell can be caused to import the reduction of efficiency because of the hydrophobic reduction of nucleic acid complexes
Deng, the amount of the signal peptide therefore imported in nucleic acid complexes can be according in the molecular weight of double chain acid molecule, target cell
Specific part or organelle, the molecular weight of signal peptide and hydrophilic etc. suitably regulate.Preferably, the N-end of signal peptide
Or C-end is incorporated into 5 '-end or the 3 '-end of double chain acid molecule.
The object lesson of the signal peptide that can be incorporated into nucleic acid carrier or double chain acid molecule is identical with above-mentioned situation, Gu Sheng
Slightly describe in detail.
(3) method importing nucleic acid in cell
The preparation of nucleic acid complexes can use arbitrary known method to carry out, for example, it is possible to by double-strandednucleic acid is divided
Son and nucleic acid carrier are dissolved in the pH buffer of regulation, and mix, and place the stipulated time at the specified temperature (such as, in room
Temperature is lower places 30 minutes) prepare.Double chain acid molecule is excessive with really with the mixing ratio (mol ratio) of nucleic acid carrier preferably the latter
Form complex on the spot.Preferably double chain acid molecule is 1:4 ~ 1:30 with the mixing ratio of nucleic acid carrier, is preferably 1:10 ~ 1:
30, it is more preferably 1:15 ~ 1:20.As required, in order to remove the nucleic acid carrier of excess, it is possible to use gel filtration chromatography,
The arbitrary known method such as the chromatography of ions, reversed phase liquid chromatography carrys out purifying nucleic acid complex.
Importing nucleic acid complexes in target cell can also use arbitrary known method to carry out.(in vitro
Vitro) in time cultivating importing nucleic acid gene in cell, for example, it is also possible to by culture medium or the culture fluid comprising target cell
Middle interpolation nucleic acid complexes, at the specified temperature cultivate the stipulated time carry out, it is also possible to use electroporation, calcium phosphate method,
The method known in other such as ultrasonic irradiation method is carried out.In vivo (in vivo) to import nucleic acid complexes in target cell permissible
Carry out by employing Formulations for systemic administration or the topical etc. of the methods such as injection, drop.The efficiency imported in cell is permissible
It is marked with fluorescently-labeled double chain acid molecule by uses such as fluorescence microscopes to carry out.
The target cell becoming nucleic acid complexes and importing object is not particularly limited, as long as can be exogenous by importing
Nucleic acid control the expression of gene, then can be using arbitrary cell as target cell.
The method of nucleic acid, nucleic acid carrier and the nucleic acid complexes of importing in cell provided by the present invention is by repairing carefully
The gene of intracellular defect or reparation or correction because suffering from the defect of Insufficient cell, Ke Yishi with aberrant gene
For treating the gene therapy etc. of disease.
Embodiment
Hereinafter, illustrate for the embodiment carried out to confirm the action effect of the present invention.
[experimental implementation]
(1) nucleic acid carrier
Shown in the nucleic acid carrier table described as follows 1 used in the present embodiment.It should be noted that the aminoacid sequence of table 1
Use single-letter to be called for short to represent.It addition, in table 1, "-C18" represent the peptide that N-end is modified, "-Cholic A " through octadecyl
Representing that N-end is combined with the peptide of cholic acid via amido link, " Ac-" represents N-acetyl group amide.
In order to nucleic acid carrier (the following sequence number shown in table 1 below1~21) compare, employ as sun from
The RNAiFect (QIAGEN K.K..) of sub-property lipid system nucleic acid carrier.
[table 1]
| Sequence number | Aminoacid sequence | Serial number |
| 1 | (RL)5 | 14 |
| 2 | (RL)6 | 15 |
| 3 | (RL)7 | 16 |
| 4 | (RL)8 | 17 |
| 5 | (RL)9 | 18 |
| 6 | (RL)10 | 19 |
| 7 | (RL)7-C18 | |
| 8 | (RL)7-Cholic A | |
| 9 | (RL)8-C18 | |
| 10 | Ac--QAKKKKLDK-(RL)7-GK-OH | 20 |
| 11 | Ac-SPQPKKKP-(RL)7-GK-OH | 21 |
| 12 | Ac-RQARRNRRRRWR-(RL)7-GK-OH | 22 |
| 13 | Ac-GPKKKRKV-(RL)7-GK-OH | 23 |
| 14 | Ac-NSAAFEDLRVLS-(RL)7-GK-OH | 24 |
| 15 | Ac-GRKKRRQRRRPPQG-(RL)7-GK-OH | 25 |
| 16 | Ac-LPPLERLTL-GGGG-(RL)7-GK-OH | 26 |
| 17 | Ac-ALQKKLEELELDE-GGGG-(RL)7-GK-OH | 27 |
| 18 | Ac-LALKLAGLDI-GGGG-(RL)7-GK-OH | 28 |
| 19 | Ac-SLEGAVSEISLRD-GGGG-(RL)7-GK-OH | 29 |
| 20 | Ac-LPVLENLTL-GGGG-(RL)7-GK-OH | 30 |
| 21 | Ac-LASLMNLGMS-GGGG-(RL)7-GK-OH | 31 |
(2) target cell
Using the Jurkat cell as acute lymphoblastic leukemia cell, as chronic casual leukaemia's
K562 cell and the Hela cell as cervical cancer cell are used as target cell.
(3) double chain acid molecule
For Jurkat cell and Hela cell, use hTERT (human telomerase reverse transcriptase: with the unlimited increasing of cancerous cell
Growing is relevant.) siRNA (21 base pair) corresponding to gene is as double chain acid molecule.Jurkat cell and Hela cell
The base sequence of the siRNA that hTERT gene is corresponding is respectively shown in serial number 32 and 33 described as follows.
5'-GGAGCAAGUUGCAAAGCAUTT-3'[serial number 32]
5'-AUGCUUUGCAACUUGCUCCTT-3'[serial number 33]
For K562 cell, using Bcr/abl (is the abl gene on No. 9 chromosomes and the bcr base on No. 22 chromosomes
Present on the DNA chimeric gene produced because of fusion i.e. Philadelphia chromosome, chronic casual leukemic Disease-causing gene, makes cell increase
Grow that abnormal signal is hyperfunction, make the unordered propagation of leukaemia.) the corresponding siRNA (21 base pair) of gene is as double-strandednucleic acid
Molecule.Shown in the base sequence serial number described as follows 34 and 35 of the siRNA used.
5'-GCAGAGUUCAAAAGCCCUUTT-3'[serial number 34]
5'-AACGGCUUUUGAACUCUGCTT-3'[serial number 35]
(4) preparation of nucleic acid complexes
SiRNA aqueous solution (ultra-pure water) the 10 μ L of 200 μMs of peptide aqueous solution (ultra-pure water) 10 μ L and 20 μMs is at room temperature mixed
Close, cultivate 30 minutes at 37 DEG C.
(5) import in target cell
The nucleic acid complexes of preparation in above-mentioned (4) is added in the culture fluid or culture medium of target cell.Addition is
Any one in 100nM, 200nM or 400nM.After adding nucleic acid complexes, cultivate the stipulated time at 37 DEG C.During for cultivating
Between, the situation of Jurkat cell and K562 cell is 24 hours, and the situation of HeLa cell is 48 hours.
(6) in target cell, import the evaluation of the efficiency of nucleic acid complexes
Evaluate as follows for importing the efficiency of nucleic acid complexes in target cell: use fluorescent labeling is marked
The double chain acid molecule of note, carries out fluorescence microscope to the target cell after the nucleic acid complexes prepared and cultivation and evaluates.
(7) evaluation of half-life
Nucleic acid is obtained multiple by the rheological parameters' change with time of the resolution ratio of the siRNA contained by the nucleic acid complexes in 10%FBS at 37 DEG C
The half-life of the siRNA contained by compound.The formaldehyde of the resolution ratio of the siRNA siRNA by separating after nucleic acid complexes is heated
Modified agarose gel electrophoresis is obtained.
(8) Cytotoxic evaluation
The cytotoxicity of nucleic acid carrier is obtained by nucleic acid carrier and the rheological parameters' change with time of the survival rate of the target cell of cultivation.
(9) research of the silencing efficiency of target gene based on RNAi
The silencing efficiency of target gene based on RNAi is studied as follows: make the cell after cultivation dissolve, and uses RT-PCR
After method carries out the amplification of target gene, with beta-actin gene as internal standard, obtain the change of expression quantitatively.
[result]
(1) structure of the nucleic acid carrier in nucleic acid complexes
For preparing the siRNA of nucleic acid complexes, by aminoacid sequence (RL)7The nucleic acid carrier formed3(the sequence in table 1
Number.The most identical.) and circular dichroism (CD) spectrogram of nucleic acid complexes that obtained by these be shown in Fig. 1.Nucleic acid carrier3Single
In the presence of Du, can observe the CD spectrogram deriving from random coil structure, and on the other hand, in nucleic acid complexes, at 217nm
Can observe the distinctive strong negative Cotton effect of beta sheet structure.From these results, nucleic acid carrier3There is Double helix knot
In the presence of the double chain acid molecule of structure, define the side chain of the arginine (R) with positive charge and be arranged in one side side, hydrophobic
The side chain of the leucine (L) of property is arranged in the beta sheet structure of side, opposite.It addition, for the different peptide of aminoacid sequence, not
Observe and double-strandednucleic acid form complex and the expression of beta sheet structure, therefore imply that: via arginic guanidine radicals with
The electrostatic interaction of phosphate and double chain acid molecule and nucleic acid carrier3Peptide chain between the one or both of hydrogen bond,
Define strong combination between the two.
(2) in target cell, import the efficiency of nucleic acid complexes
For by the alternately arranged aminoacid sequence of basic amino acid i.e. arginine and hydrophobic amino acid i.e. leucine
Row (RL)nThe nucleic acid carrier that (5≤n≤10) are formed1~6, have studied in Jurkat, import the corresponding fluorescent labeling of hTERT
The efficiency of siRNA, result is shown in Fig. 2.In order to compare, it is shown with result during RNAiFect in the lump.Can be true by these results
Recognize, during siRNA individualism, it is impossible to invade cell interior, but using RNAiFect and n is the nucleic acid carrier of more than 73~6
Time, siRNA can be imported cell interior.
Use by aminoacid sequence (RL)7The nucleic acid carrier formed3, have studied nucleic acid carrier in the lump3With siRNA mole
Comparison imports the effect that the efficiency of siRNA produces in Jurkat cell.Result is shown in Fig. 3.Be can confirm that by these results, in order to
Substantially ensure that the efficiency importing siRNA in Jurkat cell, the mol ratio of nucleic acid carrier 3 and siRNA need for more than 1:6,
More preferably more than 1:10.
To nucleic acid carrier3With4Middle importing hydrophobicity base or signal peptide (NLS) are to importing siRNA's in Jurkat cell
The effect that efficiency produces is shown in Fig. 4.Be can confirm that by these results, nucleic acid carrier3Combination (nucleic acid carrier with Jurkat cell12In), by importing octadecyl to N-end and deriving from the NLS of HIV-1Rev protein, import in Jurkat cell
The efficiency of siRNA is substantially improved.
The result tested equally for HeLa cell is shown in Fig. 5 and Fig. 6.Result according to Fig. 5 can confirm that, in order to fill
Divide and guarantee to import the efficiency of siRNA, nucleic acid carrier in HeLa cell3Need for more than 1:15 with the mol ratio of siRNA.Separately
Outward, the result of Fig. 6 can confirm that, import siRNA for HeLa cell, to nucleic acid carrier3Middle importing derives from HIV-1Rev
NES sequence (the nucleic acid carrier of protein16), derive from the NES sequence (nucleic acid carrier of Dsk-119) and derive from Matrin3
NES sequence (nucleic acid carrier21) time, the importing efficiency of siRNA increases.
(3) half-life of the siRNA contained by nucleic acid complexes
With the resolution ratio of the siRNA in gel electrophoresis monitoring 10%FBS and siRNA contained by nucleic acid complexes through time-varying
The result changed is shown in Fig. 7, Fig. 8, table 2 below and table 3.Result according to Fig. 7 can confirm that, siRNA (partly declines rapidly in 10%FBS
Phase is about 2 hours) decompose, but by with nucleic acid carrier3~6Forming nucleic acid complexes, the half-life increases to a left side, several times ~ 20 times
Right.It addition, for not observing that the nucleic acid carrier increased occurs in the efficiency importing siRNA in cell1With2, do not observe this
The increase of the half-life of sample, it can be considered that be the increase of the half-life in 10%FBS be because formed nucleic acid complexes and make
Become.It addition, for RNAiFect, do not observe the increase of the half-life caused by being formed of complex.It addition, such as Fig. 8
As shown in, import the nucleic acid carrier of NES sequence16~21Also the increase of half-life, especially nucleic acid carrier are observed16Considerable
Observe significant effect.
[table 2]
[table 3]
(4) cytotoxicity
For Jurkat cell and K562 cell, have studied at nucleic acid carrier1~6With in the presence of RNAiFect cultivate time
The rheological parameters' change with time of survival rate.Survival rate after 24 hours is shown in Fig. 9 and 10.Understand as shown in Figure 9, thin relative to Jurkat
Born of the same parents, nucleic acid carrier1~6The most all do not show cytotoxicity.On the other hand, relative to K562 cell, as shown in Figure 10, Observable
Arrive, add nucleic acid carrier with high concentration2~5Time, the survival rate after 24 hours reduces.But understand, at actually used 1 ~ 2 μM
Under the concentration of degree, nucleic acid carrier1~6All demonstrate more than 90%, cell survival rate apparently higher than RNAiFect.Especially
It is, nucleic acid carrier1With6Cytotoxicity is not almost shown relative to K562 cell.
(5) silencing efficiency of the Jurkat cell hTERT gene that the RNAi of nucleic acid complexes brings is employed
At the corresponding siRNA (21 base pairs: serial number 32) of the hTERT gene of Jurkat cell and nucleic acid carrier3's
Measurement result when cultivating Jurkat cell (48 hours) in the presence of complex, hTERT gene expression is shown in following
Table 4.Nucleic acid carrier is may validate that by this result3Demonstrate and import efficiency with RNAiFect with the nucleic acid of degree.
[table 4]
(6) silencing efficiency of the HeLa cell hTERT gene that the RNAi of nucleic acid complexes brings is employed
Answering of the corresponding siRNA (21 base pairs: serial number 33) of the hTERT gene of HeLa cell and nucleic acid carrier 3
Measurement result when cultivating HeLa cell (48 hours) in the presence of compound, hTERT gene expression is shown in following table
5.By this result it has been confirmed that in the case of nucleic acid carrier 3 is less when the addition of siRNA is than use RNAiFect, show
Go out with RNAiFect with the silencing efficiency of degree.
[table 5]
(7) silencing efficiency of the K562 cell Bcr/abl gene that the RNAi of nucleic acid complexes brings is employed
SiRNA-revNES conjugation (covalent bond body) (100nM) corresponding at the bcr/abl gene of K562 cell (has
The direct covalent bond of 5 '-end of the siRNA of the base sequence shown in serial number 34 has the aminoacid sequence shown in serial number 8
The N-end of HIV-1rev albumen NES sequence.) and nucleic acid carrier3(10 equivalent) or nucleic acid carrier
K562 cell (48 hours) is cultivated in the presence of the complex of LRALLRALLRALLRALLRALLRAL (serial number 36,10 equivalent)
Time, the measurement result of the expression of bcr/abl gene be shown in following table 6.
Be can confirm that by these results, in the case of K562 cell, by using in conjunction with the siRNA system having signal peptide
Standby nucleic acid carrier, the silencing efficiency of the efficiency and K562 cell Bcr/abl gene that import nucleic acid in cell is substantially improved.
[table 6]
Claims (18)
1. the method importing nucleic acid in cell, it is characterised in that the method possesses following operation: have arginine with
(RL) that hydrophobic amino acid leucine is alternately arrangedn(LR)nIn any one shown aminoacid sequence have double
In the presence of the double chain acid molecule of helical structure, make to have to be formed and there is the described arginic side chain of positive charge be arranged in list
Side, face, the leucic side chain of described hydrophobic amino acid are arranged in the nucleic acid carrier of the peptide chain of the beta sheet structure of side, opposite
Contact with described double chain acid molecule, via the described arginic side chain in the described peptide chain defining beta sheet structure and phosphorus
Hydrogen bond between electrostatic interaction and described double chain acid molecule and the described peptide chain defining beta sheet structure of acidic group
One or both, make described double chain acid molecule and described peptide chain combination form the operation of nucleic acid complexes；And, by institute
State nucleic acid complexes and import the operation of cell；
Wherein, at described (RL)n(LR)nIn, R, L represent arginine residues, leucine residue respectively, and n represents more than 7 and 10
Following natural number.
The method importing nucleic acid in cell the most according to claim 1, it is characterised in that be combined at described formation nucleic acid
In the operation of thing, described double chain acid molecule is 1:4～1:30 with the mol ratio of nucleic acid carrier.
The method importing nucleic acid in cell the most according to claim 1, it is characterised in that described double chain acid molecule is
siRNA。
The method importing nucleic acid in cell the most according to any one of claim 1 to 3, it is characterised in that described core
Acid complex has the activity of the localization to target cell and/or its specific part.
The method importing nucleic acid in cell the most according to claim 4, it is characterised in that contained by described nucleic acid complexes
Described double chain acid molecule or being combined with at least partially of described nucleic acid carrier have target cell and/or its specific part
Localization activity signal peptide.
The method importing nucleic acid in cell the most according to claim 5, it is characterised in that described signal peptide is following
Any one shown aminoacid sequence in serial number 3 and serial number 8～serial number 11,
7. according to the method importing nucleic acid in cell described in claim 5 or 6, it is characterised in that the end of described signal peptide
End is incorporated into the peptide chain contained by described nucleic acid carrier or the end of described double-strandednucleic acid.
The method importing nucleic acid in cell the most according to claim 1, it is characterised in that described peptide chain has use (RL)n
The aminoacid sequence represented, and described double chain acid molecule contained by described nucleic acid complexes or described nucleic acid carrier are at least
A part is combined with the signal peptide with the activity of the localization to target cell and/or its specific part；
Wherein, R, L represent arginine residues, leucine residue respectively, and n represents the natural number of more than 7 and less than 10.
The method importing nucleic acid in cell the most according to claim 8, it is characterised in that described signal peptide is following
Any one shown aminoacid sequence in serial number 3 and serial number 8～serial number 11,
The method importing nucleic acid in cell the most according to claim 8 or claim 9, it is characterised in that the end of described signal peptide
End is incorporated into the peptide chain contained by described nucleic acid carrier or the end of described double-strandednucleic acid.
11. 1 kinds of nucleic acid complexes, it is characterised in that this nucleic acid complexes is for having arginine and the bright ammonia of hydrophobic amino acid
(RL) that acid is alternately arrangedn(LR)nIn any one shown aminoacid sequence there is double-stranded double-strand core
In the presence of acid molecule, make to have to be formed and there is the described arginic side chain of positive charge be arranged in one side side, described hydrophobic
The leucic side chain of acidic amino acid is arranged in nucleic acid carrier and the double chain acid molecule of the peptide chain of the beta sheet structure of side, opposite
Via the described arginic side chain contained in the peptide chain defining beta sheet structure of described nucleic acid carrier and described double-strand core
The electrostatic interaction of the phosphate of acid molecule and described double chain acid molecule and the described peptide chain defining beta sheet structure
Between the one or both and combining of hydrogen bond obtain；
Wherein, at described (RL)n(LR)nIn, R, L represent arginine residues, leucine residue respectively, and n represents more than 7 and 10
Following natural number.
12. nucleic acid complexes according to claim 11, it is characterised in that described double chain acid molecule is siRNA.
13. according to the nucleic acid complexes described in claim 11 or 12, it is characterised in that described double chain acid molecule or be incorporated into
Being combined with at least partially of its described nucleic acid carrier has the letter that the localization to target cell and/or its specific part is active
Number peptide.
14. nucleic acid complexes according to claim 13, it is characterised in that described signal peptide be following serial number 3 with
And any one shown aminoacid sequence in serial number 8～serial number 11,
15. nucleic acid complexes according to claim 13, it is characterised in that the end of described signal peptide is incorporated into described core
Peptide chain contained by acid vectors or the end of described double-strandednucleic acid.
16. nucleic acid complexes according to claim 11, it is characterised in that described peptide chain has use (RL)nThe amino represented
Acid sequence, and described double chain acid molecule contained by described nucleic acid complexes or at least some of combination of described nucleic acid carrier
There is the signal peptide with the activity of the localization to target cell and/or its specific part；
Wherein, R, L represent arginine residues, leucine residue respectively, and n represents the natural number of more than 7 and less than 10.
17. nucleic acid complexes according to claim 16, it is characterised in that described signal peptide is following serial number 3 He
Any one shown aminoacid sequence in serial number 8～serial number 11,
18. according to the nucleic acid complexes described in claim 16 or 17, it is characterised in that the end of described signal peptide is incorporated into institute
State the peptide chain contained by nucleic acid carrier or the end of described double-strandednucleic acid.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010-157367 | 2010-07-10 | ||
| JP2010157367 | 2010-07-10 | ||
| PCT/JP2011/065612 WO2012008361A1 (en) | 2010-07-10 | 2011-07-07 | Method for introducing nucleic acid to cell and nucleic acid complex |
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| Publication Number | Publication Date |
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| CN103180440A CN103180440A (en) | 2013-06-26 |
| CN103180440B true CN103180440B (en) | 2016-12-14 |
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