JP2019118307A - Method of transporting cell membrane penetrating peptide, construct and cargo molecules into cells - Google Patents
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本発明は、細胞膜透過性を有するペプチドに関し、具体的にはsiRNA等の核酸を効率的に細胞内に輸送できる細胞膜透過性を有するペプチドに関する。また、そのペプチドとカーゴ分子とからなる構築物に関する。更に、カーゴ分子を細胞内に輸送する方法に関する。 The present invention relates to a peptide having cell membrane permeability, and more particularly to a peptide having cell membrane permeability capable of efficiently transporting a nucleic acid such as siRNA into cells. It also relates to a construct consisting of the peptide and a cargo molecule. Furthermore, it relates to a method of transporting cargo molecules into cells.
細胞透過性を有するペプチド(以下、細胞膜透過ペプチド(Cell-Penetrating Peptides;CPPs)と略することがある。)を用いて、細胞内にsiRNA等の核酸を導入する手法が注目されている。細胞内に導入したいsiRNA等の核酸にCPPsを細胞内導入ベクターとして化学的に結合させるか、又は、遺伝子工学的にCPPsと導入したいsiRNA等の核酸との融合タンパク質を調製し、細胞培養液に混合することで、効率よく細胞内に目的分子が導入される。 A method of introducing a nucleic acid such as siRNA into cells using a peptide having cell permeability (hereinafter sometimes abbreviated as cell-penetrating peptides (CPPs)) has attracted attention. The CPPs are chemically linked as a intracellular transfer vector to a nucleic acid such as siRNA to be introduced into cells, or a fusion protein of a CPPs and a nucleic acid such as siRNA to be introduced is genetically engineered to prepare a cell culture solution. By mixing, the target molecule is efficiently introduced into the cell.
CPPsとして実際に用いられている代表的なものとして、(1)アルギニン等の塩基性アミノ酸に富むもの、(2)塩基性部分と疎水性部分を有する両親媒性ペプチド、(3)疎水性配列に若干の塩基性配列を含むペプチド、(4)疎水性ペプチド等が挙げられる。 As representative ones actually used as CPPs, (1) rich in basic amino acids such as arginine, (2) amphiphilic peptides having a basic part and a hydrophobic part, (3) hydrophobic sequence And peptides containing some basic sequences, (4) hydrophobic peptides and the like.
ペプチドベクターは様々な物質の導入に有効であるが、siRNA等の核酸の導入には不向きである場合がある。体内でsiRNAを安定に保持・保護する一方、ひとたび標的細胞に到達すればこれを速やかに放出するという、いわば相反する二つの機能を高次に満足するペプチドベクターは実現が困難である。 Peptide vectors are effective for introducing various substances, but may not be suitable for introducing nucleic acids such as siRNA. It is difficult to realize a peptide vector that satisfies the two contradictory functions, so to speak, in a manner of stably holding and protecting siRNA in the body, and rapidly releasing it once it reaches a target cell.
非特許文献１、非特許文献２及び非特許文献３には、効率的な細胞内導入を可能とするCPPsが検討されており、CPPsの膜透過におけるカチオン性官能基の重要性や、オリゴアルギニンのペプチドヘリカル構造が細胞膜透過性の向上に寄与すること等が記載されている。 Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 3 discuss CPPs that enable efficient intracellular introduction, and the importance of the cationic functional group in membrane permeation of CPPs, and oligoarginine It has been described that the peptide helical structure of (1) contributes to the improvement of cell membrane permeability.
非特許文献４には、ロイシンとAibからなる短鎖ペプチド(Leu-Leu-Aib)3をヘリカルプロモータとしてノナアルギニン(Arg)9とコンジュゲートした細胞膜透過ペプチドF-βAla-(Arg)9-(Gly)3-(L-Leu-L-Leu-Aib)3-NH2が記載されている。しかし、この細胞膜透過ペプチドは細胞毒性が強いため、標的細胞に対するsiRNAの導入は確認されていない。 Non-Patent Document 4, cell membranes short peptide (Leu-Leu-Aib) 3 consisting of leucine and Aib and nona-arginine (Arg) 9 conjugated as helical promoter penetrating peptide F-βAla- (Arg) 9 - ( Gly) 3- (L-Leu-L-Leu-Aib) 3 -NH 2 is described. However, the introduction of siRNA to target cells has not been confirmed because this cell membrane penetrating peptide is highly cytotoxic.
本発明は、siRNA等の核酸の効率的な細胞内導入を可能とする細胞膜透過ペプチドを提供することを目的とする。 An object of the present invention is to provide a cell membrane-penetrating peptide that enables efficient intracellular introduction of nucleic acid such as siRNA.
本発明にかかる細胞膜透過ペプチドは、細胞膜透過性を有する下記の式(I)で表されるペプチドである。
F-(L-Leu-L-Leu-Aib)l-(Gly)m-(Arg)n-NH2・・・(I)
式中、lは３〜５のいずれかの整数であり、ｍは０〜３のいずれかの整数であり、ｎは８〜１０のいずれかの整数であり、Fは、リンカーを介して又は介さないで、ペプチドのＮ末端に結合した蛍光標識としてのフルオロセイン化合物である。
The cell membrane penetrating peptide according to the present invention is a peptide represented by the following formula (I) having cell membrane permeability.
F- (L-Leu-L-Leu-Aib) l- (Gly) m- (Arg) n- NH 2 (I)
In the formula, l is an integer of 3 to 5, m is an integer of 0 to 3, n is an integer of 8 to 10, and F is a linker or A fluorocein compound as a fluorescent label attached to the N-terminus of the peptide without any intervening.
本発明によれば、効率的な細胞内導入を可能とする細胞膜透過ペプチドが得られる。 According to the present invention, a cell membrane permeable peptide capable of efficient intracellular introduction is obtained.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, which are for the purpose of facilitating the understanding of the principle of the present invention, and the scope of the present invention is as follows: The present invention is not limited to the embodiment, and other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.
本発明者らは、細胞膜透過性を有するオリゴアルギニン(Arg)nに対し、ヘリカル構造の形成を誘起する疎水性型ヘリカルテンプレート配列H-(L-Leu-L-Leu-Aib)l-NH2を連結させたBlockペプチドをデザインした。オリゴアルギニン(Arg)nに疎水性型ヘリカルテンプレート配列を連結させることで、アルギニン配列がヘリカル構造を形成し、両親媒性を獲得すると考えられる。 The present inventors made a hydrophobic type helical template sequence H- (L-Leu-L-Leu-Aib) 1 -NH 2 that induces formation of a helical structure to oligoarginine (Arg) n having cell membrane permeability. We designed a block peptide linked to By linking a hydrophobic type helical template sequence to oligoarginine (Arg) n , it is considered that the arginine sequence forms a helical structure and acquires amphipathicity.
本発明にかかる細胞膜透過ペプチドは、下記式(I)で示される。 The cell membrane penetrating peptide according to the present invention is represented by the following formula (I).
F-(L-Leu-L-Leu-Aib)l-(Gly)m-(Arg)n-NH2・・・(I)
式中、lは３〜５のいずれかの整数であり、ｍは０〜３のいずれかの整数であり、ｎは８〜１０のいずれかの整数である。Fは、リンカーを介して又は介さないで、ペプチドのＮ末端に結合した蛍光標識としてのフルオロセイン化合物である。式(I)で示される細胞膜透過ペプチドは、細胞毒性をほとんど示さず、しかも、細胞培養液内でsiRNAを安定に保持する一方、標的細胞内に到達すれば速やかに放出する特性を有するため、効率的なsiRNAの細胞内導入と遺伝子発現抑制が可能である。
F- (L-Leu-L-Leu-Aib) l- (Gly) m- (Arg) n- NH 2 (I)
In the formula, l is an integer of 3 to 5, m is an integer of 0 to 3, and n is an integer of 8 to 10. F is a fluorescein compound as a fluorescent label attached to the N-terminus of the peptide, with or without a linker. The cell membrane-penetrating peptide represented by the formula (I) exhibits almost no cytotoxicity and, moreover, has the property of stably retaining the siRNA in the cell culture solution and rapidly releasing it when reaching the target cell. Efficient intracellular delivery of siRNA and suppression of gene expression are possible.
Fは、リンカーを介した蛍光標識である場合が好ましく、リンカーとしては例えばβ-Ala等がある。蛍光標識としてはフルオレセイン化合物である限り、特に限定されるものではなく、例えば下記に示す化合物が挙げられる。好ましくは5-FAM若しくは6-FAM又はこれらの混合物である。 F is preferably a fluorescent label via a linker, and the linker includes, for example, β-Ala. The fluorescent label is not particularly limited as long as it is a fluorescein compound, and examples thereof include the compounds shown below. Preferably, it is 5-FAM or 6-FAM or a mixture thereof.
下記に本実施形態にかかる細胞膜透過ペプチドの好適な場合の構造式を示す。下記式においてRは例えばOHである。 The structural formula of the suitable case of the cell membrane permeation peptide concerning this embodiment is shown below. In the following formula, R is, for example, OH.
式(I)において、lは３が好ましく、ｍは３が好ましく、ｎは９が好ましい。この最も好ましい本実施形態にかかる細胞膜透過ペプチドの構造式を示す。 In formula (I), 1 is preferably 3, m is preferably 3, and n is preferably 9. 1 shows a structural formula of a cell membrane-penetrating peptide according to this most preferred embodiment.
本実施形態にかかる構築物は、本実施形態にかかる細胞膜透過ペプチドと、細胞内に輸送すべきカーゴ分子とを含む。細胞膜透過ペプチドとカーゴ分子とは共有結合的又は非共有結合的に結合する。 The construct according to the present embodiment comprises the cell membrane penetrating peptide according to the present embodiment and a cargo molecule to be transported into cells. The cell membrane penetrating peptide and the cargo molecule are linked covalently or noncovalently.
カーゴ分子は、特に限定されるものではないが、例えば、核酸、タンパク質、薬剤、又は、ナノ粒子の何れかであり、好ましくは核酸である。 The cargo molecule is not particularly limited, and is, for example, any of a nucleic acid, a protein, a drug or a nanoparticle, preferably a nucleic acid.
カーゴ分子である核酸は、ポリヌクレオチドでもオリゴヌクレオチドでもよく、DNAでもRNA分子でもよい。DNAの場合、プラスミドDNA、cDNA、ゲノミックDNA又は合成DNAでもよい。DNA及びRNAは２本鎖でも１本鎖でもよい。カーゴ分子は2本鎖RNAであるsiRNAが特に好ましい。 The nucleic acid that is a cargo molecule may be a polynucleotide or an oligonucleotide, and may be a DNA or an RNA molecule. In the case of DNA, it may be plasmid DNA, cDNA, genomic DNA or synthetic DNA. DNA and RNA may be double stranded or single stranded. The cargo molecule is particularly preferably a double stranded RNA siRNA.
本実施形態にかかるカーゴ分子を細胞内に輸送する方法は、細胞内に輸送すべきカーゴ分子と、本実施形態にかかるペプチドとを結合させて構築物を得る工程と、その構築物を細胞に導入する工程と、を有する。 The method of transporting a cargo molecule according to this embodiment into cells comprises the steps of combining a cargo molecule to be transported into cells with the peptide according to this embodiment to obtain a construct, and introducing the construct into cells And a process.
本実施形態にかかる構築物を生体（ヒトを含む動物、特に、ヒトを含む哺乳類）に投与する方法としては、経口、注射、点眼、点鼻、経肺、皮膚を介した吸収のいずれでも良く、好ましくは注射である。例えば、本実施形態にかかる構築物を静脈注射による全身投与又は患部に注射することによる局所投与が可能である。 As a method of administering the construct according to this embodiment to a living body (animal including human, particularly mammal including human), any of oral, injection, eye drop, nasal drop, pulmonary, absorption through skin may be used. Preferably it is injection. For example, systemic administration by intravenous injection or local administration by injecting the construct according to the present embodiment into the affected area is possible.
本実施形態にかかる構築物によれば、遺伝子又はタンパク質の導入される細胞内での機能を調べることが可能である。例えば、機能を調べたい遺伝子の発現を抑制するsiRNA核酸を細胞内に導入して遺伝子の発現を抑制することによってその遺伝子の機能を調べることが可能である。 According to the construct according to this embodiment, it is possible to investigate the function in the cell into which the gene or protein is introduced. For example, it is possible to investigate the function of a gene by introducing into the cell an siRNA nucleic acid which suppresses the expression of the gene whose function is to be examined and suppressing the expression of the gene.
また本実施形態にかかる構築物によれば、細胞の性質を変えることができる。例えば、特定のmRNAを分解できるsiRNAをカーゴ分子として細胞に投与すれば、そのmRNAがコードする機能性タンパク質の発現量を低下させた細胞が得られる。 Moreover, according to the construct according to the present embodiment, the property of the cell can be changed. For example, when an siRNA capable of degrading a specific mRNA is administered to cells as a cargo molecule, cells in which the expression level of the functional protein encoded by the mRNA is reduced can be obtained.
（１）ペプチド合成
マイクロウェーブを用いたFmoc固相法により、(Arg)9、A6K、Block3〜Block9のペプチドを合成した。Block3〜Block9のペプチドは下記式(I’)で示され、F,l,m,nは下記表１で示される。5,6-FAMは5-FAMと6-FAMとのisomer mixtureである。5,6-TAMRAは5-TAMRAと6-TAMRAとのisomer mixtureである。Block3,4,5,9は本実施例にかかるペプチドである。(Arg)9、A6K、Block6,7,8は比較例にかかるペプチドである。
(1) Peptide Synthesis Peptides of (Arg) 9 , A6K, and Blocks 3 to Block 9 were synthesized by the Fmoc solid phase method using microwaves. The peptides of Block 3 to Block 9 are shown by the following formula (I ′), and F, l, m, n are shown in Table 1 below. 5,6-FAM is an isomer mixture of 5-FAM and 6-FAM. 5,6-TAMRA is an isomer mixture of 5-TAMRA and 6-TAMRA. Blocks 3, 4, 5, 9 are peptides according to the present example. (Arg) 9 , A6K, Blocks 6 , 7 and 8 are peptides according to Comparative Examples.
F-β-Ala-(L-Leu-L-Leu-Aib)l-(Gly)m-(Arg)n-NH2・・・(I’) F-β-Ala- (L-Leu-L-Leu-Aib) l- (Gly) m- (Arg) n- NH 2 (I ')
(Arg)9は下記式(II)で示されるペプチドである。 (Arg) 9 is a peptide represented by the following formula (II).
5,6-FAM-βAla-(Arg)9-NH2・・・(II)
Argは下記で示される。
5,6-FAM-βAla- (Arg) 9 -NH 2 (II)
Arg is shown below.
またA6Kのペプチドは下記式(III)で示される。Aはアラニンであり、Kはリシンである。 Moreover, the peptide of A6K is shown by following formula (III). A is alanine and K is lysine.
5,6-FAM-β-Ala-AAAAAAK-NH2・・・(III)
（２）ペプチドの二次構造解析
得られたペプチドは逆相HPLCにより精製し、MALDI-MS及びLC-TOFMSによって同定した。ペプチドの溶液状態における二次構造は20 mM PBS buffer solution (pH = 7.4)：メタノール＝１：１溶媒を用い、CDスペクトル測定(190~260 nm)によって解析を行なった。その結果、本発明にかかるblockペプチドはヘリカル構造を形成していることが示された（図１）。
5,6-FAM-β-Ala-AAAAAAK-NH 2 (III)
(2) Analysis of secondary structure of peptide The obtained peptide was purified by reverse phase HPLC and identified by MALDI-MS and LC-TOFMS. The secondary structure of the peptide in the solution state was analyzed by CD spectroscopy (190 to 260 nm) using a 20 mM PBS buffer solution (pH = 7.4): methanol = 1: 1 solvent. As a result, it was shown that the block peptide of the present invention forms a helical structure (FIG. 1).
（３）siRNAの導入
siRNA導入効果に関しては、エストロゲン受容体ERα及びアポトーシス阻害タンパク質XIAPに対する化学修飾を施したsiRNA（Stealth RNAiTM siRNA(Thermo Fisher Scientific); 25-mer）各３種をOpti-MEM(登録商標)（Thermo Fisher Scientific）上で各種ペプチドと懸濁し、10分後に細胞に添加した。XIAPやERαに対するsiRNAには標的特異性や血清中における安定性の高い修飾型siRNA（25-mer）を用いた。また、芳香族炭化水素受容体(Aryl hydrocarbon receptor; AhR)及び転写因子Notch1に対する非修飾標準siRNA（21-mer）３種もしくは２種をOpti-MEM(登録商標)（Thermo Fisher Scientific）上で各種ペプチドと懸濁し、10分後に細胞に添加した。
(3) Introduction of siRNA
Regarding the siRNA introduction effect, each of three kinds of chemically modified siRNA (Stealth RNAiTM siRNA (Thermo Fisher Scientific); 25-mer) for estrogen receptor ERα and apoptosis inhibitor protein XIAP was selected as Opti-MEM (registered trademark) (Thermo Fisher) It was suspended with various peptides on Scientific) and added to cells after 10 minutes. The modified siRNA (25-mer) with high target specificity and stability in serum was used as siRNA for XIAP and ERα. In addition, 3 or 2 types of unmodified standard siRNA (21-mer) against aromatic hydrocarbon receptor (Aryl hydrocarbon receptor; AhR) and transcription factor Notch1 on Opti-MEM (registered trademark) (Thermo Fisher Scientific) Suspended with peptide and added to cells after 10 minutes.
上述したsiRNAの塩基配列を以下に示す。なお、末端のttはオーバーハングであり、DNAのチミジン2塩基である。
XIAP siRNA#1: 5’-ACACUGGCACGAGCAGGGUUUCUUU-3’（配列番号１）
XIAP siRNA#2: 5’-GAAGGAGAUACCGUGCGGUGCUUUA-3’ （配列番号２）
XIAP siRNA#3: 5’-CCAGAAUGGUCAGUACAAAGUUGAA-3’ （配列番号３）
ERalpha siRNA#1: 5’-CGACAUGCUGCUGGCUACAUCAUCU-3’ （配列番号４）
ERalpha siRNA#2: 5’-UCACAGACACUUUGAUCCACCUGAU-3’ （配列番号５）
ERalpha siRNA#3: 5’-GACCGAAGAGGAGGGAGAAUGUUGA-3’ （配列番号６）
AhR siRNA#1: 5’-GCUCUGAAUGGCUUUGUAUtt-3’ （配列番号７）
AhR siRNA#2: 5’-GCUACCACAUCCACUCUAAtt-3’ （配列番号８）
AhR siRNA#3: 5’-CCUGUAAUCAGCCUGUAUUtt-3’ （配列番号９）
NOTCH1 siRNA#1: 5’-GCAACAGCUCCUUCCACUUtt-3’ （配列番号１０）
NOTCH1 siRNA#2: 5’-GCAUGGUGCCGAACCAAUAtt-3’ （配列番号１１）
各標的タンパク質の細胞内発現量をウェスタンブロット法により検出し、定量化することで輸送効率を評価した。
The nucleotide sequence of the above-mentioned siRNA is shown below. The end tt is an overhang, which is a thymidine 2 base of DNA.
XIAP siRNA # 1: 5'-ACACUGGCACGAGCAGGGGUUUCUU-3 '(SEQ ID NO: 1)
XIAP siRNA # 2: 5'-GAAGGAGAUACCGUGCGGUGCUUUA-3 '(SEQ ID NO: 2)
XIAP siRNA # 3: 5'-CCAGAAUGGUCAGUACAAGUUGAA-3 '(SEQ ID NO: 3)
ERalpha siRNA # 1: 5'-CGACAUGCUGCUGGCUACAUCUCU-3 '(SEQ ID NO: 4)
ERalpha siRNA # 2: 5'-UCACAGACACUUUGAUCCACCUGAU-3 '(SEQ ID NO: 5)
ERalpha siRNA # 3: 5'-GACCGAAGAGGAGGGAGAAUGUUGA-3 '(SEQ ID NO: 6)
AhR siRNA # 1: 5'-GCUCUGAAUGGCUUGUAUtt-3 '(SEQ ID NO: 7)
AhR siRNA # 2: 5'-GCUACCACAUCCAUCUCUAAtt-3 '(SEQ ID NO: 8)
AhR siRNA # 3: 5'-CCUGUAAUCAGCCUGUAUUTt-3 '(SEQ ID NO: 9)
NOTCH1 siRNA # 1: 5'-GCAACAGCUCCUUCCACUUTt-3 '(SEQ ID NO: 10)
NOTCH1 siRNA # 2: 5'-GCAUGGUGCCGAACCAAUAtt-3 '(SEQ ID NO: 11)
The intracellular expression level of each target protein was detected by Western blotting and quantified to evaluate transport efficiency.
（４）Block3の実験結果
Block3は、修飾型siRNAと共にMCF-7細胞に処理すると、siRNA配列依存的に標的タンパク質(XIAP、ERα)の発現量を減少させることが明らかになった（図2(a)）。
(4) Test results of Block 3
Block 3 was found to reduce the expression level of target protein (XIAP, ERα) depending on the siRNA sequence when treated with MCF-7 cells together with the modified siRNA (FIG. 2 (a)).
またBlock3は、標準型siRNA（化学修飾を施した修飾型siRNAに対して、化学修飾を施していないsiRNAを標準型siRNAと記載することがある。）と共にMCF-7細胞に処理しても、siRNA配列依存的に標的タンパク質(AhR、NOTCH1))の発現量を減少させることが明らかになった（図2(b)）。 Also, Block 3 can be treated with MCF-7 cells together with a standard type siRNA (an siRNA without chemical modification may be described as a standard type siRNA for chemically modified modified siRNA). It became clear that the expression level of the target protein (AhR, NOTCH1) was decreased depending on the siRNA sequence (FIG. 2 (b)).
またBlock3は、修飾型siRNAと共にHeLa細胞又はHepG2細胞に処理しても、siRNA配列依存的に標的タンパク質(XIAP)の発現量を減少させることが明らかになった（図2(c)）。 Also, it was revealed that Block3 reduced the expression level of target protein (XIAP) in a manner dependent on the siRNA sequence even when HeLa cells or HepG2 cells were treated with the modified siRNA (Fig. 2 (c)).
（５）その他のペプチドの実験結果
Block3のどの構造がsiRNA導入活性に重要であるか調べるためにその他のペプチドと活性を比較した（図3）。
(5) Experimental results of other peptides
The activity was compared with other peptides in order to determine which structure of Block 3 is important for the siRNA introduction activity (FIG. 3).
図3(a)のLane 8,10,12,14を参照するに、グリシンリンカーを持たないBlock4は、Block3と比較するとそのsiRNA導入効果はやや減弱することが明らかになった。 Referring to Lanes 8, 10, 12, and 14 in FIG. 3 (a), it was revealed that Block 4 having no glycine linker slightly reduced its siRNA introduction effect as compared to Block 3.
図3(a)のLane 2,4,6を参照するに、蛍光標識をFITCに変更したBlock5は、Block3と比較するとそのsiRNA導入効果はやや減弱することが明らかになった。図3(c)のLane 8を参照するに、蛍光標識をTAMRAに変更したBlock9は、Block3と比較するとそのsiRNA導入効果はやや減弱することが明らかになった。これらの結果から、フルオロセイン化合物は、5-FAM又は6-FAMであることがsiRNA導入効率に好ましいことが判明した。 Referring to Lanes 2, 4 and 6 in FIG. 3 (a), Block 5 in which the fluorescent label was changed to FITC was found to slightly attenuate its siRNA introduction effect as compared to Block 3. Referring to Lane 8 in FIG. 3 (c), it was revealed that Block 9 in which the fluorescent label was changed to TAMRA slightly reduced its siRNA introduction effect as compared to Block 3. From these results, it was found that the fluorescein compound is preferably 5-FAM or 6-FAM for the efficiency of siRNA introduction.
図3(c)のLane 2を参照するに、(Arg)9はXIAPの発現量を抑制できなかった。図3(c)のLane 4を参照するに、A6KはXIAPの発現量を抑制できなかった。図3(b)を参照するに、Block6,7,8はXIAPの発現量を抑制できなかった。これらの結果から、(Arg)9やヘリカルプロモータ配列単独ではsiRNA導入効果を示さず、(Arg)9に対して適切な長さ(l=3〜5)のヘリカルプロモータ配列の連結が、siRNA導入活性に必須であることが判明した。 Referring to Lane 2 of FIG. 3 (c), (Arg) 9 failed to suppress the expression level of XIAP. Referring to Lane 4 of FIG. 3 (c), A6K failed to suppress the expression level of XIAP. As shown in FIG. 3 (b), Blocks 6, 7 and 8 could not suppress the expression level of XIAP. These results are consolidated helical promoter sequences in (Arg) 9 and helical promoter sequence alone showed no siRNA introduction effect, (Arg) suitable lengths for 9 (l = 3~5), siRNA introduced It turned out to be essential for activity.
標的細胞における遺伝子機能の調査、標的細胞の改変等に利用できる。 It can be used to investigate gene function in target cells, modify target cells, etc.
配列番号１〜４：プライマー Sequence number 1-4-a primer
Claims (8)
F-(L-Leu-L-Leu-Aib)l-(Gly)m-(Arg)n-NH2・・・(I)
〔式中、
lは３〜５のいずれかの整数であり、
ｍは０〜３のいずれかの整数であり、
ｎは８〜１０のいずれかの整数であり、
Fは、リンカーを介して又は介さないで、ペプチドのＮ末端に結合した蛍光標識としてのフルオロセイン化合物である。〕で表されるペプチド。 Following formula (I) having cell membrane permeability
F- (L-Leu-L-Leu-Aib) l- (Gly) m- (Arg) n- NH 2 (I)
[In the formula,
l is an integer of 3 to 5, and
m is an integer from 0 to 3,
n is an integer of 8 to 10,
F is a fluorescein compound as a fluorescent label attached to the N-terminus of the peptide, with or without a linker. ] The peptide represented by these.
前記構築物を細胞に導入する工程と、を有する、
カーゴ分子を細胞内に輸送する方法。 Combining a cargo molecule to be transported into a cell with the peptide according to claim 1 or 2 to obtain a construct;
Introducing the construct into cells.
A method of transporting cargo molecules into cells.
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