CN1568373A - 介导rna干涉的小rna分子 - Google Patents

介导rna干涉的小rna分子 Download PDF

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CN1568373A
CN1568373A CNA018209009A CN01820900A CN1568373A CN 1568373 A CN1568373 A CN 1568373A CN A018209009 A CNA018209009 A CN A018209009A CN 01820900 A CN01820900 A CN 01820900A CN 1568373 A CN1568373 A CN 1568373A
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dsrna
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T·图舍尔
S·埃尔巴沙
W·兰德科尔
M·威尔姆
R·吕尔曼
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Abstract

双链RNA(dsRNA)在多种生物中通过一种被称作RNA干涉(RNAi)的过程诱导序列特异的转录后基因沉默。利用果蝇体外系统,我们证明了19-23 nt短RNA片段是RNAi的序列特异性介体。短干涉RNA(siRNA)通过长dsRNA的RNase III样加工反应产生。化学合成的具有突出3’端的siRNA双链体在裂解液中介导高效的靶RNA切割,切割位点位于指导siRNA所覆盖的区域的中心附近。此外,我们还证明了dsRNA的加工方向决定是有义还是反义靶RNA能被产生的siRNP复合物切割。

Description

介导RNA干涉的小RNA分子
发明领域
本发明涉及介导靶特异的核酸修饰(如RNA干涉和/或DNA甲基化)所需的双链(ds)RNA分子的序列和结构特征。
发明背景
向线虫C.elegans中注射dsRNA导致在序列上与注射的dsRNA高度同源的基因特异性沉默(Fire等人,1998),在这一发现后,提出术语“RNA干涉”(RNAi)。之后也在昆虫、青蛙(Oelgeschlager等人,2000)和包括小鼠在内的其他动物(Svoboda等人,2000;Wianny和Zernicka-Goetz,2000)中观察到RNAi,在人类中也可能存在。RNAi与植物中共抑制和真菌中抑制(quelling)的转录后基因沉默(PTGS)机制密切相关(Catalanotto等人,2000;Cogoni和Macino,1999;Dalmay等人,2000;Ketting和Plasterk,2000;Mourrain等人,2000;Smardon等人,2000),RNAi机制的一些组分也是共抑制引起的转录后沉默所必需的(Catalanotto等人,2000;Dernburg等人,2000;Ketting和Plasterk,2000)。最近也对这一主题进行了综述(Bass,2000;Bosher和Labouesse,2000;Fire,1999;Plasterk和Ketting,2000;Sharp,1999;Sijen和kooter,2000),参见2000年3月2日发行的《植物分子生物学》第43卷的完整内容。
在植物中,除了PTGS之外,导入的转基因也能通过RNA指导的胞嘧啶DNA甲基化导致转录基因沉默(参见Wassenegger,2000)。植物中短至30bp的基因组靶标以RNA指导的方式甲基化(Pelissier,2000)。DNA甲基化在哺乳动物中也存在。
RNAi和共抑制的自然功能似乎是保护基因组免受移动遗传因子(例如在有活性时在宿主细胞中产生异常RNA或dsRNA的反转录转座子和病毒)的入侵(Jensen等人,1999;Ketting等人,1999;Ratcliff等人,1999;Tabara等人,1999)。特异性mRNA降解阻止转座子和病毒的复制,尽管某些病毒通过表达抑制PTGS的蛋白质能克服或阻止这一过程(Lucy等人,2000;Voinnet等人,2000)。
dsRNA只触发与dsRNA相同的区域内的同源RNA特异性降解(Zamore等人,2000)。dsRNA被加工为21-23nt RNA片段,靶RNA切割位点规则地间隔21-23nt。因此提示21-23nt片段是靶标识别的指导RNA(Zamore等人,2000)。在细胞裂解前用dsRNA转染黑尾果蝇(D.melanogaster Schneider)2细胞,在由该细胞制备的提取物中也检测到这种短RNA(Hammond等人,2000),然而,显示序列特异性核酸活性的级分也含有相当一部分残余dsRNA。观察到从加工的dsRNA中分离的21-23nt片段在一定程度上能介导特异性mRNA降解,这进一步支持了21-23nt片段在指导mRNA切割中的作用(Zamore等人,2000)。大小相似的RNA分子也在显示PTGS的植物组织中积累(Hamilton和Baulcombe,1999)。
在此,我们利用建立的果蝇(Drosophila)体外系统(Tuschl等人,1999;Zamore等人,2000)进一步研究RNAi机制。我们证实,当短21nt和22nt RNA与3’突出端碱基配对时,它们作为序列特异性mRNA降解的指导RNA。在该系统中短30bp dsRNA不能介导RNAi,因为它们不再加工为21nt和22nt RNA。此外,我们还确定了相对于21和22nt短干涉RNA(siRNA)的靶RNA切割位点,并且证实,dsRNA加工的方向决定了是有义还是反义靶RNA能被产生的siRNP内切核酸酶复合物切割。此外,siRNA也可能是转录调节的重要工具,例如通过引导DNA甲基化使哺乳动物基因沉默。
在人类体内细胞培养系统(HeLa细胞)中进行的其它实验表明,长度优选地为19-25个核苷酸的双链RNA分子具有RNAi活性。因此,与果蝇的结果不同,24和25nt长双链DNA分子对于RNAi也是有效的。
发明内容
本发明的目的在于提供能够介导靶特异性RNA干涉或其它靶特异性核酸修饰(如DNA甲基化)的新型试剂,这种试剂与现有试剂相比具有更高的效能和安全性。
一种分离的双链RNA分子提供了这一问题的解决方案,其中每条RNA链的长度为19-25,特别是19-23个核苷酸,其中该RNA分子能够介导靶特异性核酸修饰,特别是RNA干涉和/或DNA甲基化。优选地,至少一条链具有1-5个核苷酸、更优选地1-3个核苷酸、最优选地2个核苷酸的3’-突出端。另一条链可以是平端,或者具有可达6个核苷酸的3’突出端。如果dsRNA的两条链都正好是21或22nt,当两端都是平端时(0nt突出),也能观察到一定程度的RNA干涉。该RNA分子优选地是合成RNA分子,基本上不含细胞提取物,例如果蝇胚胎中存在的污染物。此外,该RNA分子优选地基本上不含任何非靶特异的污染物,特别是非靶特异的RNA分子,例如细胞提取物中存在的污染物。
此外,本发明还涉及分离的双链RNA分子的用途,其中每条RNA链的长度为19-25个核苷酸,用于介导哺乳动物细胞中,特别是人类细胞中靶特异的核酸修饰,特别是RNAi。
惊奇地发现,合成的短双链RNA分子,特别是具有突出3’端的,是序列特异的RNAi的介体,介导高效的靶-RNA切割,其中切割位点靠近指导短RNA覆盖的区域的中心。
优选地,RNA分子的每条链的长度为20-22个核苷酸(或者在哺乳动物细胞中为20-25个核苷酸),其中每条链的长度可以相同或者不同。3’-突出端的长度优选地达到1-3个核苷酸,其中每条链的突出端长度可以相同或者不同。RNA-链优选地含有3’-羟基。5’-端优选地含有磷酸、二磷酸、三磷酸或羟基。最有效的dsRNA由配对的两条21nt链组成,使得dsRNA的两端存在1-3nt,特别是2nt的3’突出端。
siRNA指导的靶RNA切割反应是高度序列特异的。然而,不是siRNA的所有位点对于靶标识别都有相同的作用。在siRNA双链体中心的错配最为关键,基本上消除靶RNA切割。相反,与单链靶RNA互补的siRNA链的3’核苷酸(例如位点21)对于靶标识别的特异性就没有作用。此外,与靶RNA具有相同极性的siRNA链的未配对2nt 3’突出端的序列对于靶RNA切割并不重要,因为只有反义siRNA链才能指导靶标识别。因此,从单链突出核苷酸起,只有反义siRNA的倒数第二个位点(例如位点20)需要匹配靶向的有义mRNA。
令人吃惊的是,本发明的双链RNA分子在血清或用于细胞培养的生长培养基中显示很高的体内稳定性。为了进一步提高稳定性,可以稳定3’突出端使其免受降解,例如,可以加以选择,使其由嘌呤核苷酸,特别是腺苷或鸟苷核苷酸组成。此外,嘧啶核苷酸置换为修饰类似物,例如尿苷2nt 3’突出端置换为2’-脱氧胸苷可以耐受,不影响RNA干涉的效率。不含2’羟基可以显著提高突出端在组织培养基中的核酸酶抗性。
在本发明的一个特别优选的实施方案中,RNA分子可含有至少一种修饰的核苷酸类似物。该核苷酸类似物可位于基本上不影响靶特异性活性(如RNAi介导活性)的位置上,例如位于双链RNA分子的5’端和/或3’端区。特别是,掺入修饰的核苷酸类似物可以稳定突出端。
优选的核苷酸类似物选自糖修饰或骨架修饰的核糖核苷酸。然而应当指出,核碱基修饰的核糖核苷酸也是合适的,即核糖核苷酸,其含有非天然存在的核碱基代替天然存在的核碱基,如5-位修饰的尿苷或胞苷,例如5-(2-氨基)丙基尿苷、5-溴尿苷;8-位修饰的腺苷和鸟苷,例如8-溴鸟苷;去氮杂核苷酸,例如7-去氮杂-腺苷;O-和N-烷基化核苷酸,例如N6-甲基腺苷。在优选的糖修饰的核糖核苷酸中,2’OH-基被替换为选自H、OR、R、卤素、SH、SR、NH2、NHR、NR2或CN的基团,其中R是C1-C6烷基、烯基或炔基,卤素是F、Cl、Br或I。在优选的骨架修饰的核糖核苷酸中,与相邻核糖核苷酸连接的磷酯基团被替换为一个修饰的基团,例如硫代磷酸酯(phosphothioate)基。应当指出,上述修饰可以组合。
为了介导靶特异的RNAi和/或DNA甲基化,本发明的双链RNA分子的序列必须与核酸靶分子具有足够的同一性。优选地,该序列与RNA分子双链部分中的希望的靶分子至少50%,特别是至少70%相同。在RNA分子的双链部分中,同一性更优选地至少85%,最优选地100%。一种双链RNA分子与预定的核酸靶分子(如mRNA靶分子)的同一性可以如下确定:
I = - n L × 100
其中I是百分同一性,n是dsRNA的双链部分中与靶标相同的核苷酸数,L是dsRNA的双链部分与靶标的序列重叠的长度。
此外,也可包括3’突出端,特别是长度为1-3个核苷酸的突出端,确定双链RNA分子与靶序列的同一性。在此情况中,与靶序列的序列同一性优选地至少50%,更优选地至少70%,最优选地至少85%。例如,来自3’突出端的核苷酸和来自双链5’和/或3’端的可达2个核苷酸可以修饰,而活性没有显著丧失。
本发明的双链RNA分子可以用包括下列步骤的方法制备:
(a)合成两条RNA链,每条长度为19-25个,例如19-23个核苷酸,其中该RNA链能形成双链RNA分子,优选地至少一条链具有1-5个核苷酸的3’-突出端,
(b)在一定条件下结合合成的RNA链,其中形成一种双链RNA分子,它能介导靶特异的核酸修饰,特别是RNA干涉和/或DNA甲基化。
合成RNA分子的方法在本领域中众所周知。在本文中,具体是指如Verma和Eckstein(1998)所述的化学合成法。
由合成的DNA模板或者由分离自重组细菌的DNA质粒进行酶转录也能制备单链RNA。一般使用噬菌体RNA聚合酶,如T7、T3或SP6 RNA聚合酶(Milligan和Uhlenbeck(1989))。
本发明的另一个方面涉及一种介导细胞或生物中的靶特异性核酸修饰(特别是RNA干涉和/或DNA甲基化)的方法,包括下列步骤:
(a)在可以发生靶特异性核酸修饰的条件下使细胞或生物接触本发明的双链RNA分子,和
(b)将双链RNA完成的靶特异性核酸修饰导向含有基本对应于双链RNA的序列部分的靶核酸。
优选地,接触步骤(a)包括将双链RNA分子导入靶细胞中,例如分离的靶细胞,例如细胞培养物,单细胞微生物,或多细胞生物内的一个靶细胞或多个靶细胞。更优选地,该导入步骤包括载体介导的输送,例如利用脂质体载体或通过注射。
本发明的方法可用来确定细胞或生物中一种基因的功能,乃至调节细胞或生物中一种基因的功能,它能够介导RNA干涉。细胞优选地是真核细胞或细胞系,例如植物细胞或动物细胞,如哺乳动物细胞,例如胚细胞、多能干细胞、肿瘤细胞,例如畸胎癌细胞或病毒感染的细胞。生物优选地是真核生物,例如植物或动物,如哺乳动物,特别是人类。
本发明的RNA分子针对的靶基因可能与病理状态有关。例如,该基因可能是病原体相关基因,例如病毒基因,肿瘤相关基因,或自身免疫病相关基因。靶基因也可能是在重组细胞或遗传改变的生物中表达的异源基因。通过确定或调节,特别是抑制这种基因的功能,可能获得在农业领域或在医学或兽医学领域有价值的信息和治疗效益。
dsRNA通常作为药用组合物施用。可用已知方法进行施用,其中向体外或体内的希望的靶细胞中导入核酸。常用的基因转移技术包括磷酸钙、DEAE-葡聚糖、电穿孔、显微注射和病毒法(Graham,F.L.和van der Eb,A.J.(1973)Virol.52,456;McCutchan,J.H.和Pagano,J.S.(1968),J.Natl.Cancer Inst.41,351;Chu,G.等人(1987),Nucl.AcidsRes.15,1311;Fraley,R.等人(1980),J.Biol.Chem.255,10431;Capecchi,M.R.(1980),Cell 22,479)。向细胞中导入DNA的一种最新技术是使用阳离子脂质体(Felgner,P.L.等人(1987),Proc.Natl.Acad.Sci USA 84,7413)。市售的阳离子脂制剂有,例如Tfx 50(Promega)或Lipofectamin2000(Life Technologies)。
因此,本发明也涉及一种药用组合物,其含有至少一种上述双链RNA分子作为活性剂和一种药用载体。该组合物可在人类医学或兽医学中用于诊断和治疗用途。
为用于诊断或治疗,该组合物可以是溶液形式,例如可注射溶液,乳膏、软膏、片剂、悬液等。该组合物可以用任何适当的方法施用,例如注射、口服、局部、鼻、直肠给药等。载体可以是任何合适的药用载体。优选地使用能提高RNA分子进入靶细胞的效能的载体。这类载体的合适的例子有脂质体,特别是阳离子脂质体。更加优选的施用方法是注射。
RNAi法的另一个优选用途是对真核细胞或真核非人类生物的功能分析,优选的是哺乳动物细胞或生物,最优选的是人类细胞,例如细胞系,如HeLa或293,或啮齿动物,例如大鼠或小鼠。通过用与预定靶基因同源的适当双链RNA分子或编码适当双链RNA分子的DNA分子转染,能够在靶细胞,例如细胞培养物或靶生物中获得特定敲除表型。惊奇地发现,存在短双链RNA分子不会引起宿主细胞或宿主生物的干扰素反应。
因此,本发明的另一个主题是显示靶基因特异的敲除表型的真核细胞或真核非人类生物,其中包含至少一种内源靶基因的表达至少部分缺陷,其中用能够抑制至少一种内源靶基因表达的至少一种双链RNA分子或用编码能够抑制至少一种内源靶基因表达的至少一种双链RNA分子的DNA转染该细胞或生物。应当指出,由于RNAi的特异性,本发明能够靶特异性地敲除几种不同的内源基因。
细胞或非人类生物,特别是人类细胞或非人类哺乳动物的基因特异性敲除表型可以在分析方法中使用,例如复杂生理过程的功能和/或表型分析,如基因表达谱和/或蛋白质组的分析。例如,人们可以在培养的细胞中制备人类基因的敲除表型,假定它们是备选剪接过程的调节剂。这类基因具体包括SR剪接因子家族的成员,如ASF/SF2、SC35、SRp20、SRp40或SRp55。另外也能分析SR蛋白对预定的其它剪接基因(如CD44)的mRNA谱的影响。优选地利用基于寡核苷酸的芯片通过高通量方法进行这种分析。
利用基于RNAi的敲除技术,可以抑制靶细胞或靶生物中内源靶基因的表达。内源基因可以用编码靶蛋白或靶蛋白变体或突变形式的外源靶核酸(例如基因或cDNA)补充,外源核酸任选地可与编码可检测肽或多肽(例如亲和标记物,特别是多亲和标记物)的另一种核酸序列融合。靶基因的变体或突变形式不同于内源靶基因,因为由于单个或多个氨基酸的置换、插入和/或缺失,它们编码在氨基酸水平上与内源基因产物不同的基因产物。变体或突变形式可具有与内源靶基因相同的生物活性。另一方面,变异或突变的靶基因也可能具有不同于内源靶基因的生物活性,例如活性部分缺失、活性完全缺失、活性提高等。
这种互补可以如下完成:共表达外源核酸编码的多肽,例如含有靶蛋白和亲和标记物的融合蛋白和用于敲除靶细胞中内源基因的双链RNA分子。这种共表达可以利用一种合适的表达载体或者利用表达载体的组合实现,该载体表达外源核酸编码的多肽(例如标记物修饰的靶蛋白)和双链RNA分子。在靶细胞中从头合成的蛋白质和蛋白质复合物将含有外源基因产物,例如修饰的融合蛋白。为了避免RNAi双链分子抑制外源基因产物表达,编码外源核酸的核苷酸序列在与双链RNA分子同源的序列部分中可能在DNA水平上改变(在氨基酸水平上引起或不引起突变)。此外,也可以用来自其它种(例如来自小鼠)的相应核苷酸序列补充内源靶基因。
本发明的细胞或生物的优选用途是对基因表达谱和/或蛋白质组的分析。在一个特别优选的实施方案中,对一种或几种靶蛋白的变体或突变形式进行分析,其中利用如上所述的外源靶核酸将这种变体或突变形式重新导入细胞或生物中。内源基因敲除与利用突变的(例如部分缺失的)外源靶标拯救相组合比使用敲除细胞具有优势。而且,这种方法还特别适用于鉴定靶蛋白的功能域。在另一个优选实施方案中,例如对至少两种细胞或生物的基因表达谱和/或蛋白质组和/或表型特征进行比较。这些生物选自:
(i)一种对照细胞或对照生物,没有靶基因抑制,
(ii)一种细胞或生物,具有靶基因抑制,和
(iii)一种细胞或生物,具有靶基因抑制加外源靶核酸的靶基因互补。
本发明的方法和细胞也适用于鉴定和/或表征药理试剂的方法,例如从一组待测物质中鉴定新的药理试剂,和/或表征已知药理试剂的作用机制和/或副作用。
因此,本发明也涉及一种鉴定和/或表征作用于至少一种靶蛋白的药理试剂的系统,其包括:
(a)一种真核细胞或真核非人类生物,它能表达至少一种编码该靶蛋白的内源靶基因,
(b)至少一种双链RNA分子,它能抑制所述至少一种内源靶基因的表达,和
(c)一种待测物质或一组待测物质,其中将要鉴定和/或表征这种待测物质或组合的药理学性质。
如上所述的系统优选地还包括:
(d)至少一种编码靶蛋白或其变体或其突变形式的外源靶核酸,其中该外源靶核酸在核酸水平上不同于内源靶基因,因此与内源靶基因的表达相比,外源靶核酸的表达受双链RNA分子的抑制大大减少。
此外,RNA敲除互补法还可用于制备目的,例如从真核细胞,特别是哺乳动物细胞,更具体地是从人类细胞中亲和纯化蛋白质或蛋白质复合物。在本发明的实施方案中,外源靶核酸优选地编码与亲和标记物融合的靶蛋白。
该制备方法可以用于高分子量蛋白质复合物的纯化,其分子量优选地≥150kD,更优选地≥500kD,任选地可含有核酸,如RNA。具体例子有:由U4/U6 snRNP颗粒的20kD、60kD和90kD蛋白质组成的异源三聚蛋白质复合物,由分子量14、49、120、145和155kD的5种蛋白质组成的来自17S U2 snRNP的剪接因子SF3b,含有U4、U5和U6 snRNA分子和大约30种蛋白质的25S U4/U6/U5三-snRNP颗粒,其分子量约为1.7MD。
该方法适用于哺乳动物细胞,特别是人类细胞的功能蛋白质组分析。
下面,通过下列附图和实施例更详细地说明本发明。
附图说明
图1:短至38bp的双链RNA能介导RNAi。
(A)用于靶向Pp-Luc mRNA的dsRNA的图示。制备覆盖29-504bp的三个系列的平端dsRNA。标出了dsRNA有义链的第一个核苷酸相对于Pp-Luc mRNA起始密码子的位置(p1)。(B)RNA干涉测定(Tuschl等人,1999)。靶Pp-Luc与对照Rr-luc活性之比用缓冲液对照(黑条)标准化。dsRNA(5nM)在果蝇裂解液中25℃预温育15分钟,之后加入7-甲基-鸟苷-加帽的Pp-luc和Rr-luc mRNA(~50pM)。继续温育1小时,然后通过双萤光素酶测定(Promega)分析。数据是至少4次独立实验的平均值±标准差。
图2:29bp dsRNA不再加工为21-23nt片段。在果蝇裂解液中内部32P-标记的dsRNA(5nM)加工后形成21-23mer的时程。标出了dsRNA的长度和来源。RNA分子量标记(M)加于左道中,并标出片段大小。时间为0时的双条带是由于dsRNA的不完全变性。
图3:短dsRNA只切割mRNA靶标一次。
(A)10nM帽32P-标记的有义或反义RNA与p133系列的10nMdsRNA在果蝇裂解液中温育1小时,产生的稳定5’切割产物的变性凝胶电泳。帽标记的靶RNA部分核酸酶T1消化并部分碱性水解(OH)产生长度标记物。dsRNA针对的区域在两端表示为黑条。显示了111bp长dsRNA的主要切割位点之间的20-23nt间隔。水平箭头表明不是由于RNAi引起的非特异性切割。(B)有义和反义靶RNA上切割位点的位置。加帽177nt有义和180nt反义靶RNA的序列以反平行方向表示,使得互补序列彼此相对。不同dsRNA针对的区域用位于有义与反义靶序列之间的不同颜色的线条标出。切割位点用环形标出:大环为强切割,小环为弱切割。32P-放射性标记的磷酸基以星号标记。
图4:21和22nt RNA片段通过RNase III样机制产生。
(A)dsRNA加工后~21nt RNA的序列。定向克隆dsRNA加工产生的~21nt RNA片段并测序。来源于dsRNA有义链的寡核糖核苷酸用蓝线表示,来源于反义链的用红线表示。如果在多个克隆中含有相同的序列,则使用粗线,右侧的数字是指频率。dsRNA介导的靶RNA切割位点用橙色环形表示,大环表示强切割,小环表示弱切割(参见图3B)。有义链之上的环形表示有义靶标内的切割位点,dsRNA下面的环形表示反义靶标中的切割位点。在来源于dsRNA 3’端的~21nt片段中鉴定出高达5个其它核苷酸。这些核苷酸主要是C、G或A残基的随机组合,最可能是在dsRNA组成链的T7转录过程中以非模板方式添加的。(B)~21nt RNA的核苷酸组成的二维TLC分析。~21nt RNA如下产生:内部放射性标记的504bp Pp-luc dsRNA在果蝇裂解液中温育,凝胶纯化,然后用核酸酶P1(最上一行)或核糖核酸酶T2(最下一行)消化为单核苷酸。在存在指定α-32P核苷三磷酸之一的情况下,通过转录在内部放射性标记dsRNA。放射性通过磷成像检测。核苷5’-一磷酸、核苷3’-一磷酸、核苷5’,3’-二磷酸和无机磷酸盐分别表示为pN、Np、pNp和Pi。黑色环形表示非放射性载体核苷酸的UV吸收点。用T4多核苷酸激酶和γ-32P-ATP使核苷3’-一磷酸5’磷酸化,制备放射性标记标准,通过与该标准共迁移鉴定3’,5’-二磷酸(红色环形)。
图5:合成的21和22nt RNA介导靶RNA切割。
(A)对照52bp dsRNA和合成21和22nt dsRNA的图示。21和22nt短干涉RNA(siRNA)的有义链用蓝色表示,反义链用红色表示。除了双链体5的22nt反义链之外,siRNA的序列来源于52和111bpdsRNA的克隆片段(图4A)。双链体6和7中的siRNA是111bp dsRNA加工反应所特有的。在双链体1和3的合成反义链序列中存在两个3’突出核苷酸,以绿色表示。对照52bp dsRNA的两条链都通过体外转录制备,一部分转录物可能包括非模板3’核苷酸添加。siRNA双链体指导的靶RNA切割位点表示为橙色环形(见图4A的图例),如图5B所示确定。(B)有义和反义靶RNA上切割位点的位置。靶RNA序列如图3B所示。对照52bp dsRNA(10nM)或21和22nt RNA双链体1-7(100nM)与靶RNA一起在果蝇裂解液中25℃温育2.5小时。稳定的5’切割产物在凝胶上分析。切割位点在图5A中标出。52bp dsRNA针对的区域或有义或反义链在凝胶旁边以黑线标出。切割位点全都位于与dsRNA相同的区域内。为了精确确定反义链的切割位点,使用较低百分比的凝胶。
图6:短dsRNA上的长3’突出端抑制RNAi。
(A)52bp dsRNA构建体的图示。有义和反义链的3’延伸分别以蓝色和红色表示。靶RNA上的切割位点用类似于图4A的橙色环形表示,如图6B所示确定。(B)有义和反义靶RNA上切割位点的位置。靶RNA序列如图3B所示。dsRNA(10nM)与靶RNA一起在果蝇裂解液中25℃温育2.5小时。稳定的5’酶切产物在凝胶上分析。主要切割位点用水平箭头标出,也在图6A中表示。52bp dsRNA针对的区域在凝胶两侧以黑线表示。
图7:建议的RNAi模型。
预测RNAi从dsRNA(有义链为黑色,反义链为红色)主要加工为21和22nt短干扰RNA(siRNA)开始。如果dsRNA上存在短的突出3’核苷酸,它们可能有利于短dsRNA的加工。有待表征的dsRNA加工蛋白用绿色和蓝色椭圆形表示,在dsRNA上以不对称方式装配。在我们的模型中,假定的蓝色蛋白或蛋白域与siRNA链以3’-5’方向结合,而假定的绿色蛋白或蛋白域总是结合相对的siRNA链。这些蛋白质或亚组与siRNA双链体结合,并且保持其方向,这取决于dsRNA加工反应的方向。只有与蓝色蛋白结合的siRNA序列才能指导靶RNA切割。内切核酸酶复合物被称为小干扰核糖核蛋白复合物或siRNP。在此我们假定,切割dsRNA的内切核酸酶也可切割靶RNA,这可能是通过暂时置换不用于靶标识别的被动siRNA链。靶RNA然后在序列互补指导siRNA识别的区域中心切割。
图8:报道构建体和siRNA双链体。
(a)显示来自质粒pGL2-对照、pGL-3-对照和pRL-TK(Promega)的萤火虫(Pp-luc)和海肾(Rr-luc)萤光素酶报道基因区。显示SV40调节元件、HSV胸苷激酶启动子和两个内含子(线)。GL3萤光素酶的序列与GL2有95%相同,但RL与这两者完全无关。在转染的哺乳动物细胞中由pGL2表达萤光素酶比由pGL3表达大约低10倍。SiRNA双链体针对的区域在萤光素酶基因编码区下用黑线表示。(b)显示针对GL2、GL3和RL萤光素酶的siRNA双链体的有义(上)和反义(下)序列。GL2和GL3 siRNA双链体只有3个单核苷酸置换(以灰色框出)的不同。合成含有反向GL2序列的双链体invGL2作为非特异对照。2’-脱氧胸苷的2nt 3’突出端表示为TT;uGL2类似于GL2 siRNA,但是含有核糖尿苷3’突出端。
图9:siRNA双链体引起的RNA干涉。
靶与对照萤光素酶之比用缓冲液对照(bu,黑线)标准化;灰线表示Photinus pyralis(Pp-luc)GL2或GL3萤光素酶与Renillareniformis(Rr-luc)RL萤光素酶之比(左轴),白线表示RL与GL2或GL3之比(右轴)。图a、c、e、g、i描述用pGL2-对照和pRL-TK报道质粒的组合进行的实验,图b、d、f、h、j使用pGL3-对照和pRL-TK报道质粒。用于干涉实验的细胞系在每张图的顶部标出。在标准化之前及在所检测的不同细胞系之间,缓冲液对照(bu)的Pp-luc/Rr-luc比分别为:pGL2/pRL:0.5-10,pGL3/pRL:0.03-1。作图的数据是三次独立实验的平均值±S.D.。
图10:21nt siRNA、50bp和500bp dsRNA对HeLa细胞中萤光素酶表达的影响。
长dsRNA的确切长度在线条下面标出。图a、c、e描述用pGL2-对照和pRL-TK报道质粒进行的实验,图b、d、f使用pGL3-对照和pRL-TK报道质粒。数据是两次独立实验的平均值±S.D.。(a),(b)绝对Pp-luc表达,以任意发光单位作图。(c),(d)Rr-luc表达,以任意发光单位作图。(e),(f)标准化靶标与对照萤光素酶之比。siRNA双链体的萤光素酶活性之比用缓冲液对照(bu,黑线)标准化;50或500bpdsRNA的发光比分别用来自人源化GFP(hG,黑线)的50或500bpdsRNA的发光比标准化。应当指出,靶向GL2和GL3的49与484bpdsRNA之间的总体序列差异不足以使GL2与GL3靶标之间具有特异性(49bp片段中有43nt连续同一性,484bp片段中有239nt最长连续同一性)。
图11:21-nt siRNA双链体3’突出端的变异。
(A)实验策略概述。显示了加帽的和聚腺苷酸化的有义靶mRNA,并且显示了有义和反义siRNA的相对位置。根据8个不同的反义链,制备了8个系列的双链体。siRNA序列和突出核苷酸的数量以1nt的幅度改变。(B)在5nM平端dsRNA存在下,用黑尾果蝇胚胎裂解液中的对照萤光素酶(Renilla reniformis,Rr-luc)标准化的靶萤光素酶(Photinus pyralis,Pp-luc)的相对发光。在dsRNA存在下测定的发光比用缓冲液对照(bu,黑线)获得的发光比标准化。标准化的发光比小于1表明有特异性干涉。(C-J)8个系列21-nt siRNA双链体的标准化干涉比。siRNA双链体的序列在条图之上显示。每张图显示由特定反义指导siRNA和5种不同的有义siRNA形成的一组双链体的干涉比。突出核苷酸的数量(3’突出端,正数;5’突出端,负数)在x轴上标出。数据点是至少3次独立实验的平均值,误差条代表标准差。
图12:siRNA双链体有义链长度的变化。
(A)实验图示。3条21-nt反义链与8个有义siRNA配对。这些siRNA的3’端长度改变。反义siRNA的3’突出端为1-nt(B)、2-nt(C)或3-nt(D),而每个系列的有义siRNA突出端不同。标出了siRNA双链体的序列和相应的干涉比。
图13:保留2-nt 3’突出端的siRNA双链体的长度变化。
(A)实验图示。21-nt siRNA双链体在序列上与图11H或图12C所示相同。这些siRNA双链体延伸到有义siRNA的3’端(B)或有义siRNA的5’端(C)。标出了siRNA双链体的序列和各自的干涉比。
图14:siRNA核糖残基的2’-羟基的置换。
将siRNA双链体链中的2’-羟基(OH)置换为2’-脱氧(d)或2’-O-甲基(Me)。3’端的2-nt和4-nt 2’-脱氧置换分别表示为2-nt d和4-nt d。尿苷残基置换为2’-脱氧胸苷。
图15:具有2-nt 3’突出端的21-nt siRNA双链体对有义和反义靶RNA切割的作图。
(A)32P(星号)帽标记的有义和反义靶RNA和siRNA双链体的图示。有义和反义靶RNA切割的位置分别在siRNA双链体之上和之下用三角形标出。(B)靶RNA切割位点的作图。10nM靶标与100nMsiRNA双链体在黑尾果蝇胚胎裂解液中温育2小时后,在测序凝胶上分析5’帽标记的底物和5’切割产物。长度标记物通过靶RNA的部分RNase T1消化(T1)和部分碱性水解(OH-)产生。图左侧的粗线表示与靶标同向的siRNA链1和5所覆盖的区域。
图16:指导siRNA的5’端决定靶RNA切割位置。
(A,B)实验策略图示。所有siRNA双链体中的反义siRNA相同,但是通过改变3’端有义链有18-25nt的不同(A),或者通过改变5’端有18-23nt的不同(B)。有义和反义靶RNA切割位置分别在siRNA双链体之上和之下用三角形标出。(C,D)利用帽标记的有义(上图)或反义(下图)靶RNA对靶RNA切割的分析。只显示帽标记的5’切割产物。标出了siRNA双链体的序列,有义siRNA链的长度在图上部标出。图(C)中用破折号标记的对照道显示在不含siRNA情况下温育的靶RNA。标记物如图15所述。(D)下图中的箭头指出相差1nt的靶RNA切割位点。
图17:siRNA双链体3’突出端的序列变异。
2-nt 3’突出端(NN,灰色)如图所示在序列和组成上有变化(T,2’-脱氧胸苷,dG,2’-脱氧鸟苷,星号,野生型siRNA双链体)。标准化的干涉比如图11所述确定。野生型序列与图14所示相同。
图18:靶标识别的序列特异性。
显示了错配siRNA双链体的序列,修饰的序列片段或单核苷酸以灰色在下面列出。参照双链体(ref)和siRNA双链体1-7含有2’-脱氧胸苷2-nt突出端。胸苷修饰的参照双链体的沉默率与野生型序列相当(图17)。标准化的干涉比如图11所述确定。
图19:保留2-nt 3’突出端的siRNA双链体的长度变化。
siRNA双链体延伸至有义siRNA的3’侧(A)或有义siRNA的5’侧(B)。标出了siRNA双链体的序列和各自的干涉比。对于HeLa SS6细胞,靶向GL2萤光素酶的siRNA双链体(0.84μg)用pGL2-对照和pRL-TK质粒一起转染。为了进行对比,标出了在黑尾果蝇裂解液中检测到的siRNA双链体的体外RNAi活性。
实施例1
合成小RNA介导的RNA干涉
1.1.实验方法
1.1.1体外RNAi
体外RNAi和裂解液的制备如前所述进行(Tuschl等人,1999;Zamore等人,2000)。使用新溶解的肌酸激酶(Roche)对于最佳ATP再生至关重要。在25℃下,经过15分钟的延长预温育时间,用5nM的dsRNA浓度进行RNAi翻译测定(图1),之后加入体外转录、加帽并且聚腺苷酸化的Pp-luc和Rr-luc报道mRNA。继续温育1小时,利用双萤光素酶测定(Promega)和单光3010C发光计(PharMingen)分析Pp-luc和Rr-luc蛋白的相对含量。
1.1.2RNA合成
由携带T7或SP6启动子序列的PCR模板体外转录RNA使用标准方法,例如参见(Tuschl等人,1998)。合成的RNA用Expedite RNA亚磷酰胺(Proligo)制备。3’接头寡核苷酸用二甲氧基三苯甲基-1,4-苯二甲醇-琥珀酰-氨丙基-CPG合成。寡核糖核苷酸在3ml 32%氨/乙醇(3/1)中55℃去保护4小时(Expedite RNA)或55℃ 16小时(3’和5’接头DNA/RNA嵌合寡核苷酸),然后脱硅烷化,如前所述凝胶纯化(Tuschl等人,1993)。包含长3’突出端的dsRNA制品的RNA转录物由有义方向含有T7启动子、反义方向含有SP6启动子的PCR模板产生。用GCG TAATACGACTCACTATAGAACAATTGCTTTTACAG(下划线标出T7启动子)作为5’引物,用ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA(下划线标出SP6启动子)作为3’引物,用线性化Pp-luc质粒(pGEM-luc序列)(Tuschl等人,1999)作为模板,PCR扩增有义和反义靶RNA的转录模板;T7转录的有义RNA为177nt长,在相对于起始密码子的位点113-273之间含有Pp-luc序列,之后在3’端是SP6启动子序列的17nt互补序列。通过只含单启动子序列的两种不同PCR产物的转录,制备用于形成平端dsRNA的转录物。
利用苯酚/氯仿抽提进行dsRNA退火。等摩尔浓度的有义和反义RNA(50nM-10μM,取决于长度和含量)在0.3M NaOAc(pH6)中90℃温育30秒,然后在室温下用等体积的苯酚/氯仿抽提,之后用氯仿抽提除去残余的苯酚。加入2.5-3倍体积的乙醇沉淀产生的dsRNA。沉淀溶解于裂解缓冲液(100mM KCl,30mM HEPES-KOH,pH7.4,2mM Mg(OAc)2)中,在1×TAE缓冲液中通过标准琼脂糖凝胶电泳证实dsRNA的质量。具有17nt和20nt 3’突出端的52bp dsRNA(图6)在95℃下温育1分钟退火,迅速冷却到70℃,然后在3个小时内缓慢冷却到室温(50μl退火反应,1μM标准浓度,300mM NaCl,10mMTris-HCl,pH7.5)。然后用苯酚/氯仿抽提dsRNA,乙醇沉淀,溶解于裂解缓冲液中。
用1mM ATP、CTP、GTP、0.1或0.2mM UTP和0.2-0.3μM32P-UTP(3000 Ci/mmol),或者用是放射性标记核苷三磷酸的各自的比例而不是UTP的比例转录用于制备dsRNA的内部32P-放射性标记的RNA(图2和图4)。靶RNA的帽子的标记如前所述进行。靶RNA在帽子标记后凝胶纯化。
1.1.3切割位点作图
标准RNAi反应如下进行:预温育10nM dsRNA 15分钟,随后加入10nM帽子标记的靶RNA。再温育2小时(图2A)或2.5小时(图5B和6B)后,通过蛋白酶K处理终止反应(Tuschl等人,1999)。然后在8%或10%测序凝胶上分析样品。21和22nt合成RNA双链体以100nM终浓度使用(图5B)。
1.1.4~21nt RNA的克隆
在不含靶RNA的情况下,在果蝇裂解液中温育放射性标记的dsRNA,产生21nt RNA(200μl反应,1小时温育,50nM dsP111,或100nM dsP52或dsP39)。然后用蛋白酶K处理反应混合物(Tuschl等人,1999),在变性15%聚丙烯酰胺凝胶上分离dsRNA加工产物。切下大小范围至少为18-24nt的条带,洗脱到0.3M NaCl中,在4℃下在硅化试管中过夜。通过乙醇沉淀回收RNA,去磷酸化(30μl反应,30分钟,50℃,10U碱性磷酸酶,Roche)。通过苯酚/氯仿抽提终止反应,乙醇沉淀RNA。3’接头寡核苷酸(pUUUaaccgcatccttctcx:大写,RNA;小写,DNA;p,磷酸;x,4-羟甲基苄基)与去磷酸化的~21ntRNA连接(20μl反应,30分钟,37℃,5μM 3’接头,50mM Tris-HCl,pH7.6,10mM MgCl2,0.2mM ATP,0.1mg/ml乙酰BSA,15%DMSO,25U T4 RNA连接酶,Amersham-Pharmacia)(Pan和Uhlenbeck,1992)。加入等体积的8M尿素/50mM EDTA终止混合物终止连接反应,直接加样到15%凝胶上。连接产率超过50%。从凝胶中回收连接产物,并5’-磷酸化(20μl反应,30分钟,37℃,2mM ATP,5U T4多核苷酸激酶,NEB)。通过苯酚/氯仿抽提终止磷酸化反应,乙醇沉淀回收RNA。然后,5’接头(tactaatacgactcactAAA:大写,RNA;小写,DNA)如上所述与磷酸化连接产物连接。凝胶纯化新的连接产物,在用作载体的反转录引物(GACTAGCTG GAATTCAAGGATGCGGTTAAA,下划线,EcoRI位点)存在下,从凝胶切片中洗脱。反转录(15μl反应,30分钟,42℃,150U Superscript II反转录酶,Life Technologies)之后用CAGCCAACG GAATTCATACGACTCACTAAA(下划线,EcoRI位点)作为5’引物和3’RT引物进行PCR。经苯酚/氯仿抽提和乙醇沉淀纯化PCR产物。然后用EcoRI(NEB)消化PCR产物,用T4 DNA连接酶(高浓度,NEB)连环化。在低熔点琼脂糖凝胶上分离大小为200-800bp的多联体,利用标准的熔化和苯酚抽提法从凝胶中回收,乙醇沉淀。在标准条件下与Taq聚合酶72℃温育15分钟,补平未配对的末端,用TOTO TA克隆试剂盒(Invitrogen)将DNA产物与pCR2.1-TOPO载体直接连接。利用PCR和M13-20和M13反向测序引物筛选集落。直接对PCR产物进行委托测序(Sequence Laboratories GttingenGmbH,德国)。每个克隆平均获得4-5条21mer序列。
1.1.5 2D-TLC分析
放射性标记、凝胶纯化的siRNA的核酸酶P1消化和2D-TLC如(Zamore等人,2000)所述进行。采用2μg/μl载体tRNA和30U核糖核酸酶T2(Life Technologies),在10mM乙酸铵(pH4.5)中以10μl反应体积进行核酸酶T2消化,50℃ 3小时。非放射性标准的迁移通过UV影像法测定。T2消化产物与使用γ-32P-ATP和T4多核苷酸激酶经商品核苷3’-一磷酸的5’-32P-磷酸化制备的标准共迁移,由此证实核苷-3’,5’-二磷酸的身份(数据未显示)。
1.2结果与讨论
1.2.1dsRNA加工为21和22nt RNA片段的长度要求
由黑尾果蝇合胞体胚胎制备的裂解液概述了体外RNAi,提供了一个用于生化分析RNAi机制的新型工具(Tuschl等人,1999;Zamore等人,2000)。RNAi对dsRNA的长度要求的体外和体内分析揭示,在降解靶mRNA上,短dsRNA(<150bp)比长dsRNA效率更低(Caplen等人,2000;Hammond等人,2000;Ngo等人,1998;Tuschl等人,1999)。mRNA降解效率降低的原因尚不清楚。因此我们在最佳条件下,在果蝇裂解液中,检查了靶RNA降解对dsRNA的精确长度要求(Zamore等人,2000)。合成了几个系列的dsRNA,它们针对萤火虫萤光素酶(Pp-luc)报道RNA。用双萤光素酶测定(Tuschl等人,1999)监测靶RNA表达的特异性抑制(图1A和1B)。对于短至38bp的dsRNA,我们检测到靶RNA表达的特异性抑制,但是29-36bp的dsRNA在该方法中无效。此作用与靶位点无关,与dsRNA长度(即长dsRNA)相关的Pp-luc mRNA表达的抑制程度比短dsRNA更有效。
曾经提出,dsRNA加工产生的21-23nt RNA片段是RNA干涉和共抑制的介体(Hamilton和Baulcombe,1999;Hammond等人,2000;Zamore等人,2000)。因此我们分析了大小为501-29bp的dsRNA亚组的21-23nt片段形成率。对于39-501bp长dsRNA,容易地检测到果蝇裂解液中21-23nt片段的形成(图2),但是29bp dsRNA明显延迟。这一发现与21-23nt片段在指导mRNA切割上的作用一致,为RNAi缺乏30bp dsRNA提供了一种解释。21-23mer形成的长度信赖性可能反映了一种生物学相关的控制机制,其通过规则细胞RNA的分子内碱基配对的短结构阻止不希望的RNAi激活。
1.2.2 39bp dsRNA介导靶RNA在单个位点处的切割
向果蝇裂解液中加入dsRNA和5’-加帽的靶RNA导致靶RNA的序列特异性降解(Tuschl等人,1999)。靶mRNA只在与dsRNA相同的区域内切割,许多靶切割位点相隔21-23nt(Zamore等人,2000)。因此,特定dsRNA的切割位点数预计大约对应于dsRNA的长度除以21。我们对在帽子处5’放射性标记的有义和反义靶RNA上的靶切割位点作图(Zamore等人,2000)(图3A和3B)。在测序凝胶上分离稳定的5’切割产物,通过与来自靶RNA的部分RNase T1和碱性水解梯度比较,确定切割位点。
与以前的发现(Zamore等人,2000)一致,所有靶RNA切割位点都位于与dsRNA相同的区域内。有义或反义靶标只被39bp dsRNA切割一次。每个切割位点都与dsRNA覆盖的区域的5’端相距10nt(图3B)。与39bp dsRNA有相同5’端的52bp dsRNA除了位于第一个位点下游23和24nt的两个较弱切割位点之外,在有义靶标上还产生相同的切割位点,距与dsRNA相同的区域的5’端10nt。反义靶标只切割一次,与各自dsRNA覆盖的区域的5’端相距10nt。图1所示的38-49bp dsRNA的切割位点作图显示,第一个和主要的切割位点总是位于dsRNA覆盖的区域的7-10nt下游(数据未显示)。这提示,靶RNA切割点取决于dsRNA末端,可能意味着加工为21-23mer从双链体末端开始。
较长的111bp dsRNA在有义和反义靶标上的切割位点比预期的更多,大多数似乎以20-23nt的间隔聚簇(图3A和3B)。对于较短的dsRNA,在有义靶标上的第一个切割位点与dsRNA覆盖的区域的5’端相距10nt,在反义靶标上的第一个切割位点与dsRNA覆盖的区域的5’端相距9nt。还不清楚是什么导致这种混乱的切割,但是一种可能性是,较长的dsRNA不仅可以从末端开始加工,而且也可在内部加工,或者存在我们还不清楚的一些dsRNA加工的特异性决定簇。以前也注意到21-23nt间隔的不规则性(Zamore等人,2000)。为了更好地了解dsRNA加工和靶RNA识别的分子基础,我们决定分析果蝇裂解液中39、52和111bp dsRNA加工产生的21-23nt片段的序列。
1.2.3 dsRNA通过RNase III样机制加工为21和22nt RNA
为了表征21-23nt RNA片段,我们检查了该RNA片段的5’和3’端。对凝胶纯化的21-23nt RNA进行高碘酸盐氧化,随后进行β-排除,表明存在末端2’和3’羟基。21-23mer对碱性磷酸酶处理也有反应性,表明存在5’端磷酸基团。5’磷酸和3’羟基端的存在提示,dsRNA能被类似于大肠杆菌RNase III的酶活性加工(综述见Dunn,1982;Nicholson,1999;Robertson,1990;Robertson,1982)。
利用T4 RNA连接酶将3’和5’接头寡核苷酸与纯化的21-23mer连接,进行21-23nt RNA片段的定向克隆。反转录连接产物,PCR扩增,连环化,克隆并测序。从39、52和111bp dsRNA的dsRNA加工反应中测序超过220个短RNA(图4A)。我们发现下列长度分布:1%18nt,5%19nt,12%20nt,45%21nt,28%22nt,6%23nt,2%24nt。对加工片段5’端核苷酸的序列分析表明,含有5’鸟苷的寡核苷酸被低估。这种情况最可能是由于加入了T4 RNA连接酶,该酶将5’磷酸化鸟苷识别为供体寡核苷酸;在3’端未见明显的序列偏差。来源于双链体有义或反义链3’端的许多~21nt片段含有在使用T7RNA聚合酶合成RNA过程中非模板添加核苷酸产生的3’核苷酸。有意思的是,也克隆了相当数量的内源果蝇~21nt RNA,其中一些来自LTR和非LTR反转录转座子(数据未显示)。这与RNAi在转座子沉默中的作用一致。
~21nt RNA存在于覆盖完整dsRNA序列的聚簇基团中(图4A)。显然,加工反应通过产生交错的3’末端切割dsRNA,这是RNase III切割的另一个特征。对于39bp dsRNA,在每条包含突出3’端的dsRNA组成链中发现两簇~21nt RNA,而在有义和反义靶标上只检测到一个切割位点(图3A和3B)。如果~21nt片段在介导mRNA降解的复合物中作为单链指导RNA存在,可以假定至少存在两个靶切割位点,但这不是事实。这提示,~21nt RNA可能以双链形式存在于内切核酸酶复合物中,但是只有一条链能用于靶RNA识别和切割。根据~21nt双链体与核酸酶复合物结合的方向可以简单地确定~21nt链之一在靶切割中的用途。该方向取决于原始dsRNA的加工方向。
与39bp dsRNA相比,52bp和111bp dsRNA的~21mer簇尚未确定。这些簇覆盖25-30nt的区域,最可能代表~21nt双链体的几个不同的亚群,因此指导在几个邻近位点的靶切割。这些切割区仍然主要有20-23nt的间隔。决定怎样规则的dsRNA才能被加工为~21nt片段的原则尚不清楚,但是以前发现,使用尿苷能改变切割位点的大约21-23nt的间隔(Zamore等人,2000)。大肠杆菌RNase III切割dsRNA的特异性似乎主要由抗决定簇控制,即,在相对于切割位点的特定位置处排除某些特异碱基对(Zhang和Nicholson,1997)。
为了检测加工的~21nt RNA片段中存在糖-、碱基-还是帽-修饰,我们在裂解液中温育放射性标记的505bp Pp-luc dsRNA 1小时,分离~21nt产物,用P1或T2核酸酶消化为单核苷酸。然后用2D薄层层析法分析核苷酸混合物(图4B)。P1或T2消化显示,4种天然核糖核苷酸都没有修饰。我们以前曾经分析了~21nt片段中的腺苷-肌苷转化(温育2小时后),检测到程度较小(<0.7%)的脱氨基作用(Zamore等人,2000);在裂解液中温育较短时间(1小时)使肌苷部分减至几乎无法检测的水平。可切割磷酸二酯键3’的RNase T2产生核苷3’-磷酸和核苷3’,5’-二磷酸,因而表明存在5’端一磷酸。检测到全部4种核苷3’,5’-二磷酸,提示核苷酸间键的切割没有或只有较低的序列特异性。总之,~21nt片段没有修饰,是由dsRNA产生的,因此在5’端存在5’-一磷酸和3’-羟基。
1.2.4合成21和22nt RNA介导靶RNA切割
对dsRNA加工产物的分析表明,~21nt片段是通过具有RNase III切割反应所有特征的一种反应产生的(Dunn,1982;Nicholson,1999;Robertson,1990;Robertson,1982)。RNase III在dsRNA的两条链上产生两个交错的切口,形成大约2nt的3’突出端。我们化学合成了21和22nt RNA,它们在序列上与某些克隆的~21nt片段相同,并且检测了它们介导靶RNA降解的能力(图5A和5B)。21和22nt RNA双链体在裂解液中以100nM浓度温育,浓度比52bp对照dsRNA高10倍。在这种条件下,易于检测到靶RNA切割。21和22nt双链体的浓度从100nM降到10nM仍能引起靶RNA切割。然而,双链体浓度从100nM提高到1000nM不能进一步提高靶切割,这可能是由于裂解液内存在一种限制蛋白因子。
与不介导RNAi的29或30bp dsRNA不同,具有2-4nt突出3’端的21和22nt dsRNA介导靶RNA的高效降解(双链体1、3、4、6,图5A和5B)。平端21或22nt dsRNA(双链体2、5、7,图5A和5B)降解靶标的能力降低,表明突出的3’端对于RNA-蛋白质核酸酶复合物的重建至关重要。~21nt双链体与蛋白质成分的高亲和力结合可能需要单链突出端。5’端磷酸尽管在dsRNA加工后存在,但不是介导靶RNA切割所需的,在合成的短RNA中缺乏。
合成21和22nt双链体指导有义及反义靶标在短双链体覆盖的区域内切割。这是一个重要结果,因为形成两对~21nt片段簇的39bpdsRNA(图2)只切割有义或反义靶标一次而不是两次。我们提出~21nt双链体的两条链只有一条能指导靶RNA切割,核酸酶复合物中~21nt双链体的方向取决于dsRNA加工的最初方向,从而解释了这一结果。然而,向体外系统输送已经加工好的~21nt双链体可以以对称RNA双链体的两个可能方向形成活性序列-特异性核酸酶复合物。这导致有义及反义靶标在与21nt RNA双链体相同的区域内切割。
靶切割位点位于与21或22nt指导序列互补的第一个核苷酸的11或12nt下游,即切割位点靠近21或22nt RNA所覆盖的区域的中心(图4A和4B)。替换22nt双链体有义链的两个核苷酸(比较图5A中的双链体1和3)只将反义靶标的切割位点替换了两个核苷酸。替换有义和反义链的两个核苷酸使切割位点位移两个核苷酸(比较双链体1和4)。我们预测能设计一对21或22nt RNA,它们几乎在任何特定位点均能切割靶RNA。
21和22nt RNA指导的靶RNA切割的特异性似乎是强烈的,因为未检测到异常的切割位点(图5B)。然而应当指出,21和22nt RNA双链体3’突出端存在的核苷酸在底物识别方面的作用可能比靠近切割位点的核苷酸要小。这是基于以下发现:活性双链体1或3的3’突出端中的3’核苷酸(图5A)不与靶标互补。现在利用21和22nt RNA能容易地进行RNAi特异性的详细分析。
根据具有突出3’端的21和22nt RNA介导RNA干涉的证据,我们提出将~21nt RNA命名为“短干涉RNA”或siRNA,将各自的RNA-蛋白质复合物命名为“小干涉核糖核蛋白颗粒”或siRNP。
1.2.5短dsRNA上的20nt 3’突出端抑制RNAi
我们表示,平端短dsRNA似乎从dsRNA的末端开始加工。在我们对RNAi中dsRNA长度依赖性的研究过程中,我们也分析了具有17-20nt突出3’端的dsRNA,我们吃惊地发现它们还不如平端dsRNA有效。对于可达100bp的dsRNA,长3’端的抑制作用特别明确,但是更长的dsRNA就没有这么明显。根据天然凝胶分析(数据未显示),这种作用是由于dsRNA形成不好。我们检验了长突出3’端的抑制作用是否能作为将dsRNA加工只定向于短RNA双链体两端之一的工具。
我们合成了52bp模型dsRNA的4种组合:平端、只在有义链上的3’延伸、只在反义链上的3’延伸和在两条链上的双3’延伸,并在裂解液中温育后对靶RNA切割位点作图(图6A和6B)。当双链体反义链的3’端延伸时,有义靶标的第一个和主要的切割位点丢失,反之亦然,当双链体有义链的3’端延伸时,反义靶标的强切割位点丢失。两条链的3’延伸使52bp dsRNA实际上失活。~20nt 3’延伸使dsRNA失活的一种解释是单链RNA-结合蛋白的结合,这能干扰dsRNA加工因子之一在该末端的结合。该结果也与我们的模型一致,在我们的模型中,在装配的siRNP中只有siRNA双链体的一条链能指导靶RNA切割。指导RNA切割的链的方向取决于dsRNA加工反应的方向。具有3’交错末端可能有利于加工复合物的装配。在有义链3’端的阻断只允许从反义链的相对3’端开始dsRNA加工。随后产生siRNP复合物,其中只有siRNA双链体的反义链能指导有义靶RNA切割。情况相反时同样如此。
至于较长的dsRNA(≥500bp,数据未显示),长3’延伸的抑制作用不明显,这提示我们,长dsRNA也可能含有内部dsRNA加工信号,或者可能由于多种切割因子的结合而协同加工。
1.2.6 dsRNA指导的mRNA切割的模型
新的生物化学数据更新了dsRNA如何靶向mRNA进行破坏的模型(图7)。双链RNA首先加工为主要长21和22nt的短RNA双链体,其具有类似于RNase III样反应的交错3’端(Dunn,1982;Nicholson,1999;Robertson,1982)。根据加工RNA片段的21-23nt长度,曾经推测RNase III样活性可能参与RNAi(Bass,2000)。在RNase III反应产物中发现,在siRNA的末端存在5’磷酸和3’羟基,这进一步支持了这一假说(Dunn,1982;Nicholson,1999)。细菌RNase III和真核同源物——酿酒酵母中的Rnt1p和粟酒酵母中的Pac1p在核糖体RNA和snRNA和snoRNA的加工中起作用(参见,例如Chanfreau等人,2000)。
关于来自植物、动物或人类的RNase III同源物的生物化学,人们知之甚少。主要通过数据库指导的序列分析或cDNA的克隆鉴定了两个RNase III酶家族。第一个RNase III家族的代表是1327个氨基酸长的黑尾果蝇蛋白drosha(Acc.AF116572)。C端由两个RNase III和一个dsRNA结合域组成,N端功能未知。在C.elegans(Acc.AF160248)和人类(Acc.AF189011)中也发现了密切同源物(Filippov等人,2000;Wu等人,2000)。drosha样人RNase III(Wu等人,2000)最近已经克隆并表征。该基因在人类组织和细胞系中普遍表达,该蛋白质位于细胞的核和核仁中。根据反义抑制研究推断的结果,提出了该蛋白质在rRNA加工中的作用。第二类的代表是编码1822个氨基酸长的蛋白质的C.elegans基因K12H4.8(Acc.S44849)。该蛋白质含有一个N端RNA解旋酶基序,之后是2个RNase III催化域和一个dsRNA结合基序,类似于drosha RNase III家族。在粟酒酵母(Acc.Q09884)、A.thaliana(Acc.FA187317)、黑尾果蝇(Acc.AE003740)和人类(Acc.AB028449)中具有密切同源物(Filippov等人,2000;Jacobsen等人,1999;Matsuda等人,2000)。K12H4.8 RNase III/解旋酶可能是参与RNAi的候选物。
在C.elegans中进行遗传筛选鉴定了rde-1和rde-4,它们是RNAi激活所必需的,对转座子移动或共抑制没有影响(Dernburg等人,2000;Grishok等人,2000;Ketting和Plasterk,2000;Tabara等人,1999)。这引出一种假说:这些基因对于dsRNA加工非常重要,但是不参与mRNA靶降解。这两种基因的功能仍然未知,rde-1基因产物是类似于兔蛋白elF2C的蛋白质家族的一员(Tabara等人,1999),rde-4的序列还未有描述。将来对这些蛋白质的生物化学表征将揭示其分子功能。
加工为siRNA双链体似乎从平端dsRNA或具有短(1-5nt)3’突出端的dsRNA的末端开始,以大约21-23nt的幅度加工。短dsRNA上的长(~20nt)3’交错末端抑制RNAi,这可能是通过与单链RNA结合蛋白相互作用。在短dsRNA侧翼的单链区抑制RNAi,以及短30bpdsRNA缺乏siRNA形成,可以解释mRNA中常见的结构区为什么不导致RNAi激活。
不希望被理论所束缚,我们假设dsRNA加工蛋白或其亚组在加工反应后仍与siRNA双链体结合。siRNA双链体相对于这些蛋白质的方向决定两条互补链的哪一条在指导靶RNA降解中起作用。化学合成的siRNA双链体指导有义及反义靶RNA的切割,因为它们能以两种可能的方向之一与蛋白质成分结合。
合成21和22nt siRNA双链体能用于有效的mRNA降解,这一重要发现为功能基因组学以及生物医学研究中基因表达的序列特异性调节提供了新的工具。在由于PKR反应激活而不能使用长dsRNA的哺乳动物系统中,siRNA可能有效(Clemens,1997)。因此,siRNA双链体是反义或核酶治疗剂的一种新的备选剂。
实施例2
人组织培养物中的RNA干涉
2.1方法
2.1.1RNA制备
用Expedite RNA亚磷酰胺和胸苷亚磷酰胺(Proligo,德国)化学合成21nt RNA。合成的寡核苷酸去保护并凝胶纯化(实施例1),之后经Sep-Pak C18柱(Waters,Milford,MA,USA)纯化(Tuschl,1993)。针对GL2(Acc.X65324)和GL3萤光素酶(Acc.U47296)的siRNA序列对应于相对于起始密码子第一个核苷酸的编码区152-173,针对RL(Acc.AF025846)的siRNA对应于起始密码子之后的区119-129。较长的RNA用T7 RNA聚合酶由PCR产物转录,然后用凝胶和Sep-Pak纯化。49和484bp GL2或GL3 dsRNA分别对应于相对于翻译起点的位点113-161和113-596。50和501bp RL dsRNA分别对应于位点118-167和118-618。针对人源化GFP(hG)的dsRNA合成的PCR模板从pAD3(Kehlenbach,1998)中扩增,因此50和501bp hG dsRNA分别对应于相对起始密码子的位点118-167和118-618。
为使siRNA退火,20μM单链在退火缓冲液(100mM乙酸钾,30mM HEPES-KOH pH7.4,2mM乙酸镁)中90℃温育1分钟,然后37℃ 1小时。对于50和500bp dsRNA,37℃温育步骤延长过夜,退火反应分别以8.4μM和0.84μM的链浓度进行。
2.1.2细胞培养
S2细胞在25℃下在补充了10%FBS、100单位/ml青霉素和100μg/ml链霉素的Schneider’s果蝇培养基(Life Technologies)中繁殖。293、NIH/3T3、HeLa S3、COS-7细胞在37℃下在补充了10%FBS、100单位/ml青霉素和100μg/ml链霉素的Dulbecco’s改良Eagle’s培养基中培养。细胞定期传代,以保持指数生长。转染前24小时,在大约80%平铺时,哺乳动物细胞用胰蛋白酶消化,用不含抗生素的新鲜培养基1∶5稀释(1-3×105细胞/ml),转移到24孔板中(500μl/孔)。S2细胞在分裂前不用胰蛋白酶消化。如厂商对于粘附细胞系所述,用Lipofectamine 2000试剂(Life Technologies)进行转染。每孔加入配制于脂质体中的1.0μg pGL2-对照(Promega)或pGL3-对照(Promega)、0.1μg pRL-TK(Promega)和0.28μg siRNA双链体或dsRNA;终体积为每孔600μl。细胞在转染20小时后温育,此后似乎健康。然后用双萤光素酶测定(Promega)监测萤光素酶表达。在1.1μg hGFP编码pAD3和0.28μg invGL2 inGL2 siRNA共转染后,通过对哺乳动物细胞系的荧光显微镜检,测定转染率,为70-90%。报道质粒在XL-1 Blue(Stratagene)中扩增,用Qiagen EndoFree Maxi质粒试剂盒纯化。
2.2结果与讨论
为了检验siRNA是否也能在组织培养中介导RNAi,我们合成了具有对称2nt 3’突出端的21nt siRNA双链体,它们针对编码海肾(Renilla reniformis)和萤火虫(Photinus pyralis,GL2和GL3)萤光素酶两种序列变体的报道基因(图8a,b)。siRNA双链体与报道质粒组合pGL2/pRL或pGL3/pRL利用阳离子脂质体共转染黑尾果蝇Schneider S2细胞或哺乳动物细胞。在转染20小时后测定萤光素酶活性。在检测的所有细胞系中,我们在同源siRNA双链体存在下观察到报道基因表达特异性减少(图9a-j)。显然,绝对萤光素酶表达水平不受非同源siRNA影响,表明21nt RNA双链体没有有害的副作用(对于HeLa细胞,例如图10a-d)。在黑尾果蝇Schneider S2细胞中(图9a,b),萤光素酶完全特异性抑制。在报道基因表达强50-100倍的哺乳动物细胞中,特异性抑制不完全(图9c-j)。同源siRNA致使GL2表达减少3-12倍,GL3表达减少9-25倍,RL表达减少1-3倍。对于293细胞,RLsiRNA对RL萤光素酶的靶向无效,但是GL2和GL3靶特异性反应(图9i,j)。293细胞中没有RL表达减少可能是由于其表达比检测的其它任何哺乳动物细胞系高5-20倍,和/或由于RNA二级结构或结合蛋白质,靶序列的可接近性受限。然而,同源siRNA双链体对GL2和GL3萤光素酶的特异靶向表明RNAi在293细胞中也起作用。
除了uGL2之外,所有siRNA双链体的2nt 3’突出端都由(2’-脱氧)胸苷组成。3’突出端中尿苷置换为胸苷在黑尾果蝇体外系统中良好耐受,突出端序列对靶标识别而言并不重要。选择胸苷突出端,因为推断它能提高组织培养基中和转染细胞内siRNA的核酸酶抗性。实际上,在检测的所有细胞系中,胸苷修饰的GL2 siRNA略强于未修饰的uGL2 siRNA(图9a,c,e,g,i)。可以想象,进一步修饰3’突出端核苷酸可能对siRNA双链体的输送和稳定性有其它好处。
在共转染实验中,对于组织培养基的终体积,使用25nM siRNA双链体(图9,10)。siRNA浓度提高为100nM不能提高特异性沉默作用,但是由于质粒DNA与siRNA之间脂质体包封的竞争,开始影响转染率(数据未显示)。siRNA浓度降至1.5nM不会降低特异性沉默作用(数据未显示),即使siRNA只比DNA质粒浓缩2-20倍。这表明,siRNA是介导基因沉默的非常有效的试剂,在浓度比常规反义或核酶基因靶向实验使用的浓度低几个数量级时,siRNA是有效的。
为了监测较长dsRNA对哺乳动物细胞的作用,制备了与报道基因同源的50和500bp dsRNA。用来自人源化GFP(hG)的dsRNA作为非特异性对照(Kehlenbach,1998)。当dsRNA与siRNA双链体含量相同地(不浓缩)共转染时,报道基因的表达强烈且非特异性地降低。以HeLa细胞为代表性实例说明了这一结果(图10a-d)。50bp dsRNA共转染使绝对萤光素酶活性非特异地降低10-20倍,500bp dsRNA共转染使其降低20-200倍。对于COS-7和NIH/3T3细胞也观察到类似的非特异性作用。对于293细胞,只有用500bp dsRNA才观察到10-20倍非特异性降低。>30bp dsRNA使报道基因表达非特异性降低被认为是干扰素反应的一部分。
奇怪的是,尽管报道基因表达强烈地非特异性降低,但我们可重复地检测到其它序列特异的、dsRNA介导的沉默。然而,只有在相对报道基因活性用hG dsRNA对照标准化时,特异性沉默作用才明显(图10e,f)。在检测的其它3种哺乳动物细胞系中也观察到同源dsRNA引起的2-10倍的特异性降低(数据未显示)。dsRNA(356-1662bp)的特异性沉默作用以前在CHO-K1细胞中有报道,但是检测2-4倍特异性降低所需的dsRNA的量比我们的实验大约高20倍(Ui-Tei,2000)。CHO-K1细胞的干扰素反应似乎也有缺陷。在另一篇报告中,利用萤光素酶/lacZ报道分子组合和829bp特异性lacZ或717bp非特异性GFP dsRNA检测293、NIH/3T3和BHK-21细胞的RNAi(Caplen,2000)。在这种情况下无法检测RNAi可能是由于萤光素酶/lacZ报道分子测定法的低敏感性和靶标与对照dsRNA的长度差异。我们的结果综合起来表明,RNAi在哺乳动物细胞中有活性,但是,如果干扰素系统被>30bp的dsRNA激活,则沉默作用难以检测。
总之,我们第一次在哺乳动物细胞中证明了siRNA介导的基因沉默。短siRNA的应用在人类组织培养的基因功能灭活和基因特异治疗剂的研制中具有广阔前景。
实施例3
RNA干涉对基因表达的特异性抑制
3.1材料与方法
3.1.1RNA制备和RNAi测定
化学RNA合成、退火和基于萤光素酶的RNAi测定如实施例1或2或以前的公开文献所述进行(Tuschl等人,1999;Zamore等人,2000)。所有siRNA双链体都针对萤火虫萤光素酶,萤光素酶mRNA序列来源于如(Tuschl等人,1999)所述的pGEM-luc(GenBank acc.X65316)。在加入mRNA之前,siRNA双链体在黑尾果蝇RNAi/翻译反应液中温育15分钟。基于翻译的RNAi测定至少一式三份进行。
为了对有义靶RNA切割作图,产生177-nt转录物,其对应于在相对于起始密码子的位点113-273之间的萤火虫萤光素酶序列,随后为SP6启动子序列的17-nt互补序列。为了对反义靶RNA切割作图,由模板产生166-nt转录物,它是利用5’引物TAATACGACTCACTATAGAGCCCATATCGTTTCATA(下划线标出T7启动子)和3’引物AGAGGATGGAACCGCTGG由质粒序列PCR扩增的。靶序列对应于在相对于起始密码子的位点50-215之间的萤火虫萤光素酶序列的互补序列。鸟苷酰基转移酶标记如前所述进行(Zamore等人,2000)。为了对靶RNA切割作图,在标准条件下(Zamore等人,2000),100nM siRNA双链体与5-10nM靶RNA在黑尾果蝇胚胎裂解液中25℃温育2小时。加入8倍体积的蛋白酶K缓冲液(200mMTris-Hcl pH7.5,25mM EDTA,300mM NaCl,2%w/v十二烷基硫酸钠)终止反应。加入蛋白酶K(E.M.Merck,溶于水)至终浓度为0.6mg/ml。反应液然后在65℃下温育15分钟,用苯酚/氯仿/异戊醇(25∶24∶1)抽提,用3倍体积的乙醇沉淀。样品置于6%测序凝胶上。长度标准通过帽标记的有义或反义靶RNA的部分RNase T1消化和部分碱水解产生。
3.2结果
3.2.1 21nt siRNA双链体中3’突出端的变异
如上所述,位于siRNA双链体3’末端的2个或3个未配对核苷酸在靶RNA降解上比相应的平端双链体更有效。为了更全面地分析末端核苷酸的功能,我们合成了5种21nt有义siRNA,每一个均用相对于靶RNA的一个核苷酸展示,而且合成了8种21nt反义siRNA,每一个均用相对于靶标的一个核苷酸展示(图11A)。通过组合有义和反义siRNA,产生8个系列的siRNA双链体,它们具有合成的突出末端,覆盖7nt 3’突出端到4nt 5’突出端的范围。用双萤光素酶测定系统测定siRNA双链体的干涉(Tuschl等人,1999;Zamore等人,2000)。siRNA双链体针对萤火虫萤光素酶mRNA,海肾萤光素酶mRNA作为内部控制。在siRNA双链体存在下测定靶标与对照萤光素酶活性的发光比,用不含dsRNA时的发光比标准化。为了比较,在图11B中显示长dsRNA(39-504bp)的干涉比。长dsRNA在5nM浓度下(图11A),siRNA双链体在100nM浓度下(图11C-J)测定干涉比。选择100nM的siRNA浓度,因为5nM 504bp dsRNA的完全加工将产生120nM总siRNA双链体。
21nt siRNA双链体介导RNAi的能力依赖于突出核苷酸或形成的碱基对的数量。含有4-6个3’突出核苷酸的双链体不能介导RNAi(图11C-F),含有2个或2个以上5’突出核苷酸的双链体同样不能(图11G-J)。具有2nt 3’突出端的双链体在介导RNA干涉上最有效,但是沉默率也是序列依赖性的,具有2nt 3’突出端的不同siRNA双链体观察到高达12倍的差异(比较图11D-H)。具有平端、1nt 5’突出端或1-3nt 3’突出端的双链体有时有功能。具有7nt 3’突出端的siRNA双链体所观察到的较小的沉默作用(图11C)可能是由于长3’突出端的反义作用,而不是由于RNAi。长dsRNA(图11B)与最有效的21nt siRNA双链体(图11E,G,H)之间RNAi效率的比较表明,100nM浓度的单siRNA双链体能与5nM 504bp dsRNA一样有效。
3.2.2与恒定21-nt反义siRNA配对的有义siRNA的长度变化
为了研究siRNA长度对RNAi的影响,我们制备了3个系列的siRNA双链体,使3种21nt反义链与8种18-25nt有义链结合。在每个siRNA双链体系列中,反义siRNA的3’突出端固定为1、2或3nt,而有义siRNA的3’端不同(图12A)。我们发现,与有义siRNA的长度无关,具有反义siRNA的2-nt 3’突出端的双链体(图12C)比具有1-nt或3-nt 3’突出端的双链体更有活性(图12B,D)。在具有反义siRNA的1-nt 3’突出端的第一个系列中,含有21-nt和22-nt有义siRNA的双链体(它们分别携带有义siRNA的1-nt和2-nt 3’突出端)最具活性。含有19-25nt有义siRNA的双链体也能介导RNA,但程度较低。类似地,在具有反义siRNA的2-nt突出端的第二个系列中,具有2-nt 3’突出端的21-nt siRNA双链体最具活性,其他任何与18-25nt有义siRNA的结合都有显著的活性。在具有3-nt反义siRNA 3’突出端的最后一个系列中,只有含20-nt有义siRNA和2-nt有义3’突出端的双链体能减少靶RNA表达。这些结果综合起来表明,siRNA的长度以及3’突出端的长度非常重要,具有2-nt 3’突出端的21-nt siRNA双链体最适于RNAi。
3.2.3具有恒定2-nt 3’突出端的siRNA双链体的长度变化
然后我们检查了通过保留对称2-nt 3’突出端同时改变两条siRNA链长度的影响(图13A)。制备两个系列的siRNA双链体,包括图11H的21-nt siRNA双链体作为参照。通过在有义siRNA的3’端(图13B)或在反义siRNA的3’端(图13C)延长碱基配对的片段,双链体的长度在20-25bp间不等。20-23bp双链体引起靶萤光素酶活性的特异性抑制,但是21-nt siRNA双链体比其它任何双链体有效性至少高8倍。24-和25-nt siRNA双链体不引起任何可检测的干涉。由于双链体两端的变化产生类似的作用,序列特异的影响较小。
3.2.4 2’-脱氧和2’-O-甲基修饰的siRNA双链体
为了评估siRNA核糖残基对于RNAi的重要性,检测了含有21-ntsiRNA和2-nt 3’突出端以及2’-脱氧或2’-O-甲基修饰的链的双链体(图14)。2-nt 3’突出端置换为2’-脱氧核苷酸没有影响,甚至置换配对区内与突出端相邻的另外两个核糖核苷酸产生具有明显活性的siRNA。因此,siRNA双链体的42nt中的8nt置换为DNA残基,活性没有损失。然而,一条或两条siRNA链完全置换为2’-脱氧残基消除了RNAi,置换为2’-O-甲基残基同样如此。
3.2.5靶RNA切割位点的确定
以前确定了22-nt siRNA双链体和21-nt/22-nt双链体的靶RNA切割位点。发现靶RNA切割位点位于siRNA双链体覆盖的区域的中心,在与21-或22-nt siRNA指导序列互补的第一个核苷酸的11或12nt下游。具有2-nt 3’突出端的5种不同21-nt siRNA双链体(图15A)与5’帽标记的有义或反义靶RNA在黑尾果蝇裂解液中温育(Tuschl等人,1999;Zamore等人,2000)。5’切割产物在测序凝胶上分析(图15B)。切割的有义靶RNA的量与通过基于翻译的实验测定的siRNA双链体的效率有关,siRNA双链体1、2、4(图15B和11H,G,E)切割靶RNA比双链体3和5更快(图15B和11F,D)。显然,5’切割产物和输入靶RNA的总放射性随着时间的增长不是恒定的,而且5’切割产物不积累。推测起来,由于缺乏5’-帽子的任一条poly(A)尾,切割产物一从siRNA-内切核酸酶复合物上释放,即快速降解。
有义和反义靶RNA的切割位点都位于siRNA双链体覆盖的区域的中间。根据双链体沿靶序列的1-nt置换来看,5种不同双链体产生的每种靶标的切割位点有1-nt不同。靶标精确地在靶位点11nt下游切割,靶位点与序列互补的指导siRNA的3’-核苷酸互补(图15A,B)。
为了确定是指导siRNA的5’端还是3’端控制靶RNA切割,我们设计了实验策略,在图16A和B中概述。一种21-nt反义siRNA,在该研究中保持不变,与5’或3’端修饰的有义siRNA配对。有义和反义靶RNA的切割位点如上所述确定。有义siRNA的3’端的改变(监测1-nt5’突出端到6-nt 3’突出端)不影响有义或反义靶RNA的切割位点(图16C)。有义siRNA的5’端的改变不影响有义靶RNA的切割(图16D,上图),预计是因为反义siRNA未改变。然而,反义靶RNA切割受到影响,并且强烈依赖于有义siRNA的5’端(图16D,下图)。当有义siRNA大小为20或21nt时,只切割反义靶标,切割位点时相差1-nt,表明靶标识别的siRNA的5’端控制靶RNA的切割。当从与指导siRNA的5’核苷酸配对的靶核苷酸开始以下游方向计数时,该位点位于核苷酸10与11之间(参见图15A)。
3.2.6序列效应和3’突出端的2’-脱氧置换
对于siRNA的功能,2-nt 3’突出端是优选的。我们希望了解突出核苷酸的序列对于靶标识别是否有作用,或者这是否只是内切核酸酶复合物(RISC或siRNP)重建所需的一个特征。我们合成了有义和反义siRNA,其具有AA、CC、GG、UU和UG 3’突出端,包括2’-脱氧修饰TdG和TT。野生型siRNA在有义3’突出端中含有AA,在反义3’突出端中含有UG(AA/UG)。所有siRNA双链体在干涉测定中都起作用,使靶表达至少降低5倍(图17)。最有效的siRNA双链体是序列型NN/UG、NN/UU、NN、TdG和NN/TT(N,任何核苷酸),使靶表达降低10倍以上。含有AA、CC或GG的反义siRNA 3’突出端的siRNA双链体比野生型序列UG或突变UU活性低2-4倍。这种RNAi效率的降低可能是由于倒数第二个3’核苷酸对序列特异性靶标识别的作用,因为3’端核苷酸由G变为U没有影响。
有义siRNA 3’突出端序列的改变没有显示任何依赖序列的作用,这是预料到的,因为有义siRNA对于有义靶mRNA的识别不一定有作用。
3.2.7靶标识别的序列特异性
为了检验靶标识别的序列特异性,我们向siRNA双链体的配对片段内引入序列改变,并测定沉默率。通过颠倒3-或4-nt长的短片段或点突变,引入序列改变(图18)。为了避免干扰碱基配对的siRNA双链体结构,一条siRNA链的序列改变在互补siRNA链中补偿。为了降低合成成本,所有2-nt 3’突出端序列都是TT(T,2’-脱氧胸苷)。TT/TT参照siRNA双链体在RNAi方面与野生型siRNA双链体AA/UG相当(图17)。用基于翻译的发光测定法定量介导报道mRNA破坏的能力。显示含有反向序列片段的siRNA双链体靶向萤火虫萤光素酶报道分子的能力显著降低(图18)。位于反义siRNA 3’端与中心之间的序列改变完全消除了靶RNA识别,但是靠近反义siRNA 5’端的突变显示程度较低的沉默。与预测的靶RNA切割位点正对的A/U碱基对颠换,或者远离预测位点的一个核苷酸的颠换,阻止靶RNA的切割,因此表明,siRNA双链体中心的单突变区别错配的靶标。
3.3讨论
siRNA是不仅在昆虫细胞中,而且在哺乳动物细胞中,用于灭活基因表达的有价值的试剂,具有广阔的治疗应用前景。我们系统分析了在黑尾果蝇胚胎裂解液中促进高效靶RNA降解所需的siRNA双链体的结构决定簇,从而提出了最有效的siRNA双链体的设计原则。完美的siRNA双链体能够沉默基因表达,假定使用含量相当的总RNA,其效率相当于500bp dsRNA。
3.4siRNA使用指南
高效沉默的siRNA双链体优选地由21-nt反义siRNA组成,应当选择它们形成具有2-nt 3’突出端的19bp双螺旋。2-nt 3’突出核糖核苷酸的2’-脱氧置换不影响RNAi,但是有助于降低RNA合成的成本,并且可提高siRNA双链体的RNAse抗性。然而,更广泛的2’-脱氧或2’-O-甲基修饰降低了siRNA介导RNAi的能力,这可能是通过干扰SiRNAP装配的蛋白质结合。
靶标识别是一个高度序列特异性的过程,由与靶标互补的siRNA介导。指导siRNA的3’-核苷酸对于靶标识别的特异性没有作用,而3’突出端的倒数第二个核苷酸影响靶RNA切割,错配使RNAi降低2-4倍。指导siRNA的5’端似乎也比3’端更能容许错配的靶RNA识别。位于靶RNA切割位点对面的siRNA中心的核苷酸是重要的特异性决定簇,甚至单核苷酸改变也能使RNAi降至不可检测的水平。这提示,在基因靶向实验中,siRNA双链体能区别突变的或多态性等位基因,这可能成为未来治疗发展的重要特征。
当有义和反义siRNA与内切核酸酶复合物或其定型复合物的蛋白质成分结合时,认为两者起不同的作用;该复合物中siRNA双链体的相对方向决定了哪条链能用于靶标识别。合成siRNA双链体在双螺旋结构上二重对称,但序列不对称。siRNA双链体在黑尾果蝇裂解液中与RNAi蛋白的结合将形成两种不对称复合物。在这种假定的复合物中,有义和反义siRNA的手性环境不同,因此功能不同。这种预测显然不适用于回文siRNA序列,或者能结合为同型二聚体的RNAi蛋白。为了使可影响有义与反义靶向siRNP之比的序列效应达到最小,我们建议使用含有相同3’突出序列的siRNA序列。我们推荐将有义siRNA突出端的序列调节为反义3’突出端的序列,因为有义siRNA在一般的敲除实验中没有靶标。有义和反义切割的siRNP重建的不对称性可能(部分地)导致RNAi效率的改变,用该研究使用的具有2-nt 3’突出端的多种21-nt siRNA双链体观察到这种改变(图14)。此外,靶位点处的核苷酸序列和/或靶RNA结构的可接近性可能导致这些siRNA双链体的效率的改变。
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Claims (47)

1.分离的双链RNA分子,其中每条RNA链的长度为19-25个核苷酸,该RNA分子能够靶特异性核酸修饰。
2.权利要求1的RNA分子,其中至少一条链具有1-5个核苷酸的3’-突出端。
3.权利要求1或2的RNA分子,它能靶特异性RNA干涉和/或DNA甲基化。
4.权利要求1-3中任一项的RNA分子,其中每条链的长度为19-23个,特别是20-22个核苷酸。
5.权利要求2-4中任一项的RNA分子,其中3’-突出端为1-3个核苷酸。
6.权利要求2-5中任一项的RNA分子,其中为避免降解稳定3’-突出端。
7.权利要求1-6中任一项的RNA分子,它含有至少一个修饰的核苷酸类似物。
8.权利要求7的RNA分子,其中修饰的核苷酸类似物选自糖修饰或骨架修饰的核糖核苷酸。
9.根据权利要求7或8的RNA分子,其中该核苷酸类似物是糖修饰的核糖核苷酸,2’-OH基被置换为选自H、OR、R、卤素、SH、SR1、NH2、NHR、NR2或CN的基团,其中R是C1-C6烷基、烯基或炔基,卤素是F、Cl、Br或I。
10.权利要求7或8的RNA分子,其中该核苷酸类似物是含有硫代磷酸酯基的骨架修饰的核糖核苷酸。
11.权利要求1-10中任一项的RNA分子,其序列与预定的mRNA靶分子有至少50%的同一性。
12.权利要求11的RNA分子,其中同一性至少为70%。
13.一种制备权利要求1-12中任一项的双链RNA分子的方法,包括下列步骤:
(a)合成两条RNA链,每条长度为19-25个核苷酸,其中该RNA链能够形成双链RNA分子,
(b)在一定条件下结合合成的RNA链,其中形成一种双链RNA分子,它能靶特异性核酸修饰。
14.权利要求13的方法,其中化学合成RNA链。
15.权利要求13的方法,其中酶促合成RNA链。
16.一种介导细胞或生物中靶特异性核酸修饰的方法,包括下列步骤:
(a)在可发生靶特异性核酸修饰的条件下使细胞或生物接触权利要求1-12中任一项的双链RNA分子,和
(b)将双链RNA完成的靶特异性核酸修饰导向含有基本对应于该双链RNA的序列部分的靶核酸。
17.权利要求16的方法,其中核酸修饰是RNA干涉和/或DNA甲基化。
18.权利要求16和17的方法,其中所述接触包括向可发生靶特异性核酸修饰的靶细胞中导入所述双链RNA分子。
19.权利要求18的方法,其中这种导入包括载体介导的输送或注射。
20.权利要求16-19中任一项的方法的用途,用于确定细胞或生物中一种基因的功能。
21.权利要求16-19中任一项的方法的用途,用于调节细胞或生物中一种基因的功能。
22.权利要求20或21的用途,其中该基因与一种病理状态有关。
23.权利要求22的用途,其中该基因是一种病原体相关基因。
24.权利要求23的用途,其中该基因是一种病毒基因。
25.权利要求22的用途,其中该基因是一种肿瘤相关基因。
26.权利要求22的用途,其中该基因是一种自身免疫病相关基因。
27.含有至少一种权利要求1-12中任一项的双链RNA分子作为活性剂和一种药用载体的药用组合物。
28.用于诊断的权利要求27的组合物。
29.用于治疗的权利要求27的组合物。
30.一种显示靶基因特异性敲除表型的真核细胞或真核非人类生物,其中用至少一种能够抑制内源靶基因表达的双链RNA分子或用编码能够抑制至少一种内源靶基因表达的至少一种双链RNA分子的DNA转染该细胞或生物。
31.权利要求30的细胞或生物,它是一种哺乳动物细胞。
32.权利要求31的细胞或生物,它是一种人类细胞。
33.权利要求30-32中任一项的细胞或生物,其用至少一种编码靶蛋白或靶蛋白变体或突变形式的外源靶核酸进一步转染,其中该外源靶核酸在核酸水平上不同于内源靶基因,因此与内源靶基因的表达相比,外源靶核酸的表达受双链RNA分子的抑制大大减少。
34.权利要求33的细胞或生物,其中外源靶核酸与编码可检测肽或多肽的另一种核酸序列融合。
35.权利要求30-34中任一项的细胞或生物在分析方法中的用途。
36.权利要求35在基因表达谱分析中的用途。
37.权利要求35在蛋白质组分析中的用途。
38.权利要求35-37中任一项的用途,其中对外源靶核酸编码的靶蛋白的变体或突变形式进行分析。
39.权利要求38在鉴定靶蛋白功能域中的用途。
40.权利要求35-39中任一项的用途,其中对至少两种细胞或生物进行比较,它们选自:
(i)一种对照细胞或对照生物,没有靶基因抑制,
(ii)一种细胞或生物,具有靶基因抑制,和
(iii)一种细胞或生物,具有靶基因抑制加外源靶核酸对靶基因的互补。
41.权利要求35-40中任一项的用途,其中的分析包括功能和/或表型分析。
42.权利要求30-34中任一项的细胞在制备方法中的用途。
43.权利要求41的用途,用于从真核细胞中分离蛋白质或蛋白质复合物。
44.权利要求43的用途,用于分离任选地含有核酸的高分子量蛋白质复合物。
45.权利要求35-44中任一项在鉴定和/或表征药理试剂中的用途。
46.一种系统,用于鉴定和/或表征作用于至少一种靶蛋白的药理试剂,其包括:
(a)一种真核细胞或真核非人类生物,它能表达至少一种编码所述至少一种靶蛋白的内源靶基因,
(b)至少一种双链RNA分子,它能抑制所述至少一种内源靶基因的表达,和
(c)一种待测物质或一组待测物质,其中将要鉴定和/或表征这种待测物质或组合的药理学性质。
47.权利要求46的系统,其进一步包括:
(d)编码靶蛋白或靶蛋白变体或突变形式的至少一种外源靶核酸,其中外源靶核酸在核酸水平上不同于内源靶基因,因此与内源靶基因的表达相比,外源靶核酸的表达受双链RNA分子的抑制大大减少。
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