CN103937794B - 一种增强基因表达的激活型siRNA - Google Patents
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Abstract
本发明公开了一种通过靶向具有TATA-box基序的基因启动子而特异性增强基因表达的激活型siRNA。所述的激活型siRNA具有以下特征:(1)与以TATA-box基序为中心的位置互补;(2)反义链的前两个碱基使用UA;(3)长度为23个核苷酸;(4)反义链的3’端进行甲基化修饰;(5)避免反义链3’端的错配。本发明提供的激活型siRNA可以长时间、显著地增强目标的表达。本发明在生物技术及基因治疗药物开发等领域具有很大应用价值和广阔的应用前景。
Description
技术领域
本发明属于分子生物学及生物医药技术领域,更具体地,涉及一种靶向具有TATA-box基序的基因启动子而特异性地增强基因表达的激活型siRNA。
背景技术
植物和动物的基因组中有大量的基因表达非编码RNA(non-codingRNA,ncRNA),如小干扰RNA(smallinterferingRNA,siRNA)、微小RNA(microRNA,miRNA)、核糖体RNA(ribosomalRNA,rRNA)和转运RNA(transferRNA,tRNA)等。非编码RNA在基因的转录、转录后水平乃至动物发育和疾病发生的过程中起到了重要的调节作用。利用与目的基因同源的双链RNA分子抑制目的基因表达的RNA干扰(RNAinterference,RNAi)技术在功能基因组研究、基因治疗、新药开发等领域具有广泛的应用,为治疗癌症、遗传病等疾病开辟了新的途径。
外源性siRNA主要由转基因转录或病毒感染诱导产生,内源性siRNA的来源比较广泛,如基因组上的重复序列(着丝粒、转座子)、假基因衍生的反义转录物等。siRNA的前体dsRNA出核后,在细胞质中被dsRBP蛋白识别并结合,并由Dicer酶切成21~25nt、3’端具有2nt悬垂、5’端具有磷酸基团的siRNA,然后被Ago蛋白结合而形成RISC,siRNA的反义链通过序列匹配使RISC与靶RNA特异性结合,Ago蛋白将靶RNA酶切一个缺口使其进一步被降解。除了介导基因沉默,近年来的研究还发现微小RNA(microRNA)和siRNA可以激活基因的表达。例如,miR-744和miR-1186能够靶向Ccnb1的启动子诱导其表达而调节小鼠细胞的扩增;靶向E-钙粘蛋白、p21、血管生长因子等基因启动子的dsRNAs可以长期诱导序列特异性的基因表达的激活。
上调某些基因的表达在科学研究及临床治疗中具有重要的价值,例如,可以通过增强糖尿病病人的胰岛素表达量而缓解其症状;可以通过增强DNA损伤修复基因的表达来延缓衰老;可以通过增强抑癌基因的表达来预防或治疗癌症等等。而目前尝试上调基因的方法主要是通过人工设计转录因子模拟物,但是与内源转录因子相同,会引起大量的下游靶基因的表达而不能单独特异性抬高某一个基因的表达,因此无法广泛应用。
鉴于RNAi技术在实验研究和临床应用中的重要作用,人们总结了设计沉默型siRNA的一些规则,但是目前对于靶向基因启动子的激活型siRNA的研究,鲜有报道。
发明内容
本发明的目的在于克服现有技术缺乏对于增强基因表达的激活型siRNA的研究的缺陷,提供一种针对靶向具有TATA-box基序的基因启动子而特异性地增强基因表达的激活型siRNA。
本发明所提供的激活型siRNA是通过靶向具有TATA-box基序的基因启动子而特异性地增强基因表达,具有以下特征:
(1)与以TATA-box基序为中心的位置互补;
(2)反义链的前两个碱基使用UA;
(3)长度为23个核苷酸;
(4)反义链的3’端进行甲基化修饰;
(5)避免反义链3’端的错配。
优选地,对所述的激活型siRNA的反义链的3’端4个核苷酸进行甲基化修饰。
本发明所述的增强基因表达的激活型siRNA还具有以下特征:
(1)靶向基因启动子的双链siRNA及单链反义RNA;
(2)长度为15~29碱基且3’端带dTdT悬垂的双链siRNA;
(3)体外合成的经过化学修饰的双链siRNA及单链反义RNA;
(4)3’端与靶标的配对至关重要的双链siRNA及单链反义RNA。
本发明所述的能够激活启动子的活性的siRNA,是针对人的17个具有TATA-box基序的的基因启动子设计的,这17个具有TATA-box基序的基因启动子来源为:人IL-2、Insulin、APOE、RHO、HIV-1、c-Myc、BCL2L12、HBB、CIRBP、L-Myc、CALCA、Z-globin、GAPD、LHB、IL-6、POMC和NPPA,其基因靶点序列按上述顺序分别为SEQIDNO:1~17,其中:
人IL-2:5’-CAGAATTAACAGTATAAATTGCATCTCTTGTT-3’SEQIDNO:1;
Insulin:5’-GGGCTCTGAGACTATAAAGCCAGCGGGGGCC-3’SEQIDNO:2;
APOE:5’-CAGGGGGAGCCCTATAATTGGACAAGTCTGG-3’SEQIDNO:3;
RHO:5’-GAGGTCACTTTATAAGGGTCTGGGGGGGTCA-3’SEQIDNO:4;
HIV-1:5’-CAGATGCTACATATAAGCAGCTGCTT-3’SEQIDNO:5;
c-Myc:5’-CGCCCACCGGCCCTTTATAATGCGAGGGTC-3’SEQIDNO:6;
BCL2L12:5’-AAAGTTGAACTAATAAAGTTTGTACGA-3’SEQIDNO:7;
HBB:5’-GGGCTGGGCATAAAAGTCAGGGCAGA-3’SEQIDNO:8;
CIRBP:5’-GGCGGAAGCGTATATAAGGCCGGGCTCGGGGA-3’SEQIDNO:9;
L-Myc:5’-CGCGACGAGATATAAGGCAGTCAGGAA-3’SEQIDNO:10;
CALCA:5’-AGCGGCGGGAATAAGAGCAGTCGCTGGC-3’SEQIDNO:11;
Z-globin:5’-CAGCTCCCTGTATATAAGGGGACCCT-3’SEQIDNO:12;
GAPD:5’-CCCCCGGTTTCTATAAATTGAGCCCGCA-3’SEQIDNO:13;
LHB:5’-CGAGGTATATAGCCAGATACACGA-3’SEQIDNO:14;
IL-6:5’-AATCTTAATAAGGTTTCCAATCA-3’SEQIDNO:15;
POMC:5’-GCAAGTATATAAGGACAGAGGA-3’SEQIDNO:16;
NPPA:5’-GGGGCTATAAAAAGAGGCGGCA-3’SEQIDNO:17。
本发明针对人的IL-2具有TATA-box基序的基因启动子,设计出一种激活型siRNA,其基因序列为5ua23Me3,具体为:
5’-CAUUAACAGUAUAAAUUGCAUUA-dTdT-3’
3’-dTdT-GuaauUGUCAUAUUUAACGUAAU-5’;
其中uaau为2’-OH甲基化修饰的碱基。
本发明的有益效果在于,本发明提供的激活型siRNA能够长时间、显著地增强目标基因的表达。在制备用于基因治疗的药物以及生命科学研究中也具有很大应用价值和广阔的应用前景。
附图说明
图1为靶向人的17个基因启动子的TATA-box基序的siRNA对靶标基因启动子活性的影响图,其中:图A-Q为针对人的17个基因启动子的TATA-box基序的不同位置设计siRNA,通过双荧光素酶质粒报告系统检测这些siRNA对靶标基因启动子活性影响的分析图。
图2为启动子TATA-box基序的不同位置的siRNA对其启动子活性的影响图,其中:图A为有效的激活型siRNA的靶点相对于TATA-box基序的位置分布图;图B为靶向IL-2启动子TATA-box基序的不同位置的siRNA对其启动子活性的影响图;图C为靶向INS(胰岛素)启动子TATA-box基序的不同位置的siRNA对其启动子活性的影响图;图D为靶向APOE(载脂蛋白E)启动子TATA-box基序的不同位置的siRNA对其启动子活性的影响图。
图3为激活型siRNA的序列特征和长度对靶标基因启动子活性的影响图,其中:图A为有效的激活型siRNA的反义链5’端前两位碱基是A/U的分布情况图;图B为靶向IL-2启动子TATA-box基序的siRNA反义链5’端的序列优化对其活性的影响图;图C为靶向IL-2启动子TATA-box基序的不同长度的siRNA对其活性的影响图。
图4为有效的激活型siRNA的错配耐受性分析图,其中:图A为靶向IL-2启动子TATA-box基序的siRNA的突变设计对其活性的影响;图B为靶向INS启动子TATA-box基序的siRNA的突变设计对其活性的影响。
图5为化学修饰对激活型siRNA的活性的影响图,其中:图A为实时荧光定量PCR检测不同化学修饰的siRNA在转染两天后对Jurkat细胞系中IL-2mRNA水平的影响图;图B为实时荧光定量PCR检测不同化学修饰的siRNA在转染四天后对Jurkat细胞中IL-2mRNA水平的影响图。
图6为经优化的siRNA对人或小鼠的CD4+T淋巴细胞中IL-2的表达的影响图,其中:图A为HEK293T细胞中使用不同剂量的经优化的siRNA对IL-2启动子活性的影响图;图B为经优化的siRNA对人的CD4+T淋巴细胞中IL-2的mRNA水平的影响图;图C为经优化的siRNA对人的CD4+T淋巴细胞中IL-2的蛋白表达的影响图;图D为经优化的siRNA对小鼠的CD4+T淋巴细胞中IL-2的mRNA水平的影响图。
具体实施方式
以下结合说明书附图和具体实施例来进一步解释本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试验方法、试剂以及设备为本技术领域常规方法、试剂和设备。
实施例一
质粒、microRNA模拟物、siRNA、单链RNA(ssRNA)的准备
将pMIR-Reporter载体(Promega)上的CMV启动子用限制性酶切替换为人IL-2启动子转录起始位点(TSS)附近的-400~+1bp段,构建成人IL-2启动子启动的荧光素酶报告载体。含有HIV-1、人INS、人APOE以及其他基因的启动子的报告载体的构建也是同样的方法。hsa-let-7i模拟物、ssRNA及相应的阴性对照从上海吉玛公司(Genepharma)购买。靶向基因启动子TATA-box基序的siRNA从广州锐博公司(Ribobio)购买。
细胞培养
Jurkat和HEK293T细胞系购自ATCC,并按其操作规范培养。用淋巴细胞分离液从健康人全血中分离得到人外周血淋巴细胞(PBMCs),再用人的CD4+TCellIsolationKitII(BD)从中分离出初始CD4+T细胞。从雌性8~10周龄BALB/c小鼠的脾脏中用小鼠的CD4+TCellIsolationKitII(BD)分离出小鼠初始CD4+T细胞。PBMCs和CD4+T细胞均用含有10%胎牛血清、50U/ml青霉素和50μg/ml链霉素的RPMI1640在5%CO2培养箱中培养。
实施例二siRNA的优化
根据不断推进的实验结果,分批次从广州锐博公司(Ribobio)购买靶向基因启动子TATA-box基序的siRNA:
第1组:根据TATA-box基序的位置设计不同的siRNA;
第2组:对靶向IL-2启动子TATA-box基序为中心位置的siRNA反义链5’端的序列进行以下优化:使其第一个碱基为U,或使其前两个碱基为UA;
第3组:在第2组siRNA序列的基础上再进行以下优化:使其序列长度分别为15、17、19、21、23、25、27或29个核苷酸;
第4组:根据图4说明,对靶向IL-2启动子TATA-box基序的siRNA进行化学修饰。
第5组:根据图5说明,分别对靶向IL-2、INS启动子TATA-box基序的siRNA进行突变设计。
第6组:将第3组和第4组的特征合并,合成具有综合特征的优化型siRNA,所有特征如下:使siRNA的序列与以TATA-box基序为中心的位置互补;使其前两个碱基为UA;使其长度为23个核苷酸;对反义链的3’端4个核苷酸进行甲基化修饰;避免反义链3’端的错配。
实施例三转染
按照产品说明书,用Lipofectamine2000(Invitrogen)将上述各组经过优化的siRNA和相应的荧光素酶报告质粒共转染进HEK293T细胞。
用LipofectamineRNAiMAX(Invitrogen)将经过优化的siRNA转染进Jurkat细胞系、人或小鼠的CD4+T细胞,使其终浓度为100~200nM,12~24小时后,用PMA(50ng/ml,Sigma)和离子霉素(1μM,Sigma)将Jurkat细胞系刺激48~72小时,用anti-CD3(1μg/ml,R&DSystems)和anti-CD28(5μg/ml,R&DSystems)将人的CD4+T细胞刺激48~72小时,用针对小鼠的anti-CD3(2μg/ml,R&DSystems)和anti-CD28(1μg/ml,R&DSystems)将小鼠的CD4+T细胞刺激48~72小时。
实施例四双荧光素酶报告分析
转染前一天向48孔板的每孔中铺2.5×104HEK293T细胞,等到每孔的细胞密度达到40~60%时,用Lipofectamine2000向每孔中转染5~10ng基因启动子启动的虫荧光素酶报告载体(FL)和2ng海肾荧光素酶载体(RL),并共转染siRNA或阴性对照(终浓度为20~50nM)。24~48小时后用Dual-Gloluciferaseassaysystem(Promega)检测双荧光素酶的活性,用RL信号值校正FL信号值。
实施例五实时定量反转录PCR分析
用TRIzol(Invitrogen)提取Jurkat和CD4+T细胞的总RNA,用PrimeScriptRTreagentKit(Takara)按照说明书进行反转录合成cDNA,接着用SYBRPremixExTaqIIKit(Takara)按照说明书使用CFX96Real-TimeSystem(Bio-Rad)进行定量PCR,用看家基因GAPDH或β-actin作为内参检测基因的mRNA水平。
实施例六蛋白印迹(Westernblot)分析
转染并刺激完成后,用裂解液[150mMNaCl,50mMTris-HCl(pH7.5),1mMEDTA,1%TritonX-100,0.5%NP-40]裂解收集的CD4+T细胞,加入SDS上样缓冲液煮沸变性后,用15%SDS-PAGE进行蛋白电泳,最后孵育抗人IL-2抗体(兔单抗,Abcam)和β-actin抗体(小鼠单抗,BD),孵育荧光二抗后用LI-COROdysseyscanner进行扫描。
如图6,本发明发现按照图1~5这些条件优化的激活型siRNA可以长时间显著地增强人或小鼠的CD4+T淋巴细胞中IL-2的表达。
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Claims (2)
1.一种增强基因表达的激活型siRNA,其特征在于,所述的激活型siRNA是通过靶向具有TATA-box基序的基因启动子而特异性地增强基因表达,具有以下特征:
(1)与以TATA-box基序为中心的位置互补;
(2)反义链的前两个碱基使用UA;
(3)长度为23个核苷酸;
(4)反义链的3’端进行甲基化修饰;
(5)避免反义链3’端的错配;
所述的激活型siRNA为针对人的IL-2具有TATA-box基序的基因启动子设计的,其序列为5ua23Me3,具体为:
5’-CAUUAACAGUAUAAAUUGCAUUA-dTdT-3’
3’-dTdT-GuaauUGUCAUAUUUAACGUAAU-5’;
其中uaau部分为2’-OH甲基化修饰的碱基。
2.如权利要求1所述的增强基因表达的激活型siRNA在制备用于基因治疗的药物以及生命科学研究中的应用。
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