现代医药生物技术概论课件

1、现代医药生物技术概论3/04/2012核酸技术及药物 Email: zhang_张必良咏蛹驼日殆狄灌逼匈炳烹祭母魔桨呆挟免赖盏汗厅申檀乃镐滑泉罢蜗苍郭-现代医药生物技术概论-现代医药生物技术概论ProteinRNADNAtranscriptiontranslationCCTGAGCCAACTATTGATGAAPEPTIDECCUGAGCCAACUAUUGAUGAA传统中心法则: DNA RNA Protein蟹酞噶自薯毯忱绥偶企掌轻拍恬滤跺遍镀糙鞍螺蔡苛斑遥骤桌扒桑祁森荡-现代医药生物技术概论-现代医药生物技术概论中心法则之例外DNAretroviruses use reverse trans

2、criptaseto replicate their genome(David Baltimore and Howard Temin) RNA virusesmRNA introns (splicing)(Philip Sharp and Richard Roberts)RNA editing (deamination of cytosineto yield uracil in mRNA)RNA interference (RNAi) a mechanismof post-transcriptional gene silencing utilizing double-stranded RNA(

3、Andrew Fire and Craig Mello)RNAs (ribozymes) can catalyze anenzymatic reaction(Thomas Cech and Sidney Altman)RNAProteinPrions are heritable proteins responsiblefor neurological infectious diseases(e.g. scrapie and mad cow) (Stanley Pruisner) Epigenetic marks, such as patterns ofDNA methylation, can

4、be inherited andprovide information other than the DNAsequence miRNA and piRNA etc.Nobel Prizes漱咕芳锁慌兰江徊兢浊闷歪捂韦辈睦鸟星淬米剑觅浙惭湍纶碘坦河攀跑营-现代医药生物技术概论-现代医药生物技术概论非编码RNA（ncRNAs）可以通过多种遗传机理调控DNA的结构、RNA的表达及蛋白质的翻译和功能，进而在细胞、组织或个体水平上影响生物体的正常生长发育。RNA越来越为人们所重视，一个崭新的RNA时代已来临。1957年Hoagland、Zamecnik及Stephenson等分离出tRNA1965年H

5、olley等测定了酵母丙氨酰tRNA的序列，获得1968年诺贝尔医学奖1977年Roberts and Sharp发现RNA splicing,荣获1993年诺贝尔医学奖1981年中科院等完成了酵母tRNA-Ala的人工全合成。这是世界上首次人工合成的一个具有完整生物活力的RNA1981年Cech等发现四膜虫rRNA的自我剪接，从而发现核酶（ribozyme）与Altman 荣获1989年诺贝尔化学奖1993年Ambros发现第一个miRNA(lin-4）1998年Fire和Mello发现RNAi,获得2006年度诺贝尔医学奖1999年Steitz等发现核糖体是核酶，获得2009年度诺贝尔化学

6、奖2000年Reinhart等发现第二个miRNA(let-7）2001年Tuschl等发现siRNA(small interfering RNA)2006年Tuschl和Hannon等发现piRNA2009年Rinn等发现LincRNARNA重大历史事件舀衔剪厕魄惩原桅矢歼眺讽翟底钉便的万宰佛柬插确浆贤匣蕉窥欲疥臀醚-现代医药生物技术概论-现代医药生物技术概论Overview of DNA to RNA to ProteinA gene is expressed in two stepsTranscription: RNA synthesisTranslation: Protein synt

7、hesis九皆吏旦蔡锦宜个涪厢鸿况祖蛀骨达接镜屿条结柜游呈问其穗黑叭欺员逸-现代医药生物技术概论-现代医药生物技术概论核糖核酸（RNA）任何序列 多种多样 2结构和功能与蛋白，代谢物和其它核酸结合功能RNAs:Riboregulators： piRNAs miRNAs siRNAs LincRNARiboswitchesRibozymes调控水平:转录翻译拘瓶楚捌减街叛毙擞胳涯队荤竿供庄沿媚蚤借腊狸玲诚骏漳芝淆荆崩嫌洞-现代医药生物技术概论-现代医药生物技术概论LincRNA调控作用LincRNA是基因间的长链非编码RNA（large intergenic noncoding RNA，简称li

8、ncRNA ） 。圆网斑洒匪苦置迟输炒顿戈涎绑廷罪按夜债洛立费谁褒丈师满部柞迪射繁-现代医药生物技术概论-现代医药生物技术概论Part I: 核酶（Ribozyme）Part II: 适配体(Aptamer)和SELEX技术Part III: 反义核酸（Antisense） Part IV: RNA干扰（RNA interference, RNAi)核酸技术及药物 孪疏娘室孤苇酷催膜站诀捆涤弧包充眩辈鹿陋苇歇拂占淹字象杏操赛妻暮-现代医药生物技术概论-现代医药生物技术概论核酸技术及药物 1. 什么是核酶（RIBOZYME）?2.核酶有哪几类?3.什么是适配体（Aptamer）?4. SELEX

9、的原理是什么?5. 核酸药物现状?6. 反义核酸治疗原理?7. Fire和Mello发现RNAi的原理是什么?8. RNAi的应用有哪些?9. RNA干扰（RNAi）技术作为治疗药物最大障碍是什么?氦寄渗犯起诺纯叶三队姿整沸打上奴扔询仍述虹兵节羚湍菊谬浑忱番欣铅-现代医药生物技术概论-现代医药生物技术概论Part I: Ribozymes(核酶)A brief HistoryHow many ribozyme ? Why ?Catalytic efficiency, condition3D structure of ribozyme:And a mechanism of catalysisAp

10、plication ?蝗侵勉圈焦憾痢蓉趁垂谍溶攻不疽躇洱肾榜氦辖苟苔京特彼咐站销际煽遂-现代医药生物技术概论-现代医药生物技术概论History of enzymatic catalysis W. Kuhne introdues the term “enzyme”1893 W. Ostwald shows that enzymes are catalysts1926 J. Summer crystallizes the enzyme urease and demonstrates that it is a protein.1977 P. Sharp discovers the splicing

11、 of introns.1982 T. Cech shows that the intron of one Tetrahymena rRNA is self splicing. He proposes the term “ribozyme” to refer to catalytic RNA1983 S. Altman demonstrates that the RNA moiety of RNAse P is sufficient for part of its activity.毒乘句嵌敞妙闻圆他脐幂晚部处蓬克壬财遵獭绊经血魄杯梧析家枪昆纫嘶-现代医药生物技术概论-现代医药生物技术概论核酶

12、(Ribozyme)由于这类酶具有类似核糖核酸酶功能，而化学本质为核酸，因此被Cech称之为核酶(Ribozyme) 。 核酶是一种具有核酸内切酶活性的RNA分子，可特异性地切割靶RNA序列，具有解离后重复切割相同靶分子的能力。耍缚窖鸽褐芜毁助毋捌层蹄从袖眠课川另臃骋喳敛缔过凰货慕庚窖戈讼回-现代医药生物技术概论-现代医药生物技术概论Thomas R.CechT.R.Cech（1947-）,因发现RNA的生物催化作用而与奥尔特曼共同获得1989年诺贝尔化学奖. 他独立地发现核糖核酸(RNA)不仅像过去所设想的那样仅被动地传递遗传信息,还起酶的作用,能催化细胞内的为生命所必需的化学反应.在他们的

13、发现之前,人们认为只有蛋白质才能起酶的作用.他最先证明RNA分子能催化化学反应,并于1982年公布其研究结果。1983年证实RNA的这种酶活动。1982: Self-splicing in Tetrahymena pre-rRNA (group I intron)Kruger et al, and Cech, Cell 31, 147-157 (1982) 痘诺动惭晌陋谎铺硷昆啃组宜媚奔讶署榜矗协块海勋添真抿掣高绘戒殊美-现代医药生物技术概论-现代医药生物技术概论Sidney Altman 奥尔特曼(S.Altman)（1939-） 因发现RNA的生物催化作用而获奖. Altman与Cech分

14、别发现了核糖核酸(RNA)自身具有的生物催化作用,这项研究不仅为探索RNA的复制能力提供了线索，而且说明了最早的生命物质是同时具有生物催化功能和遗传功能的RNA，打破了蛋白质是生物起源的定论。1983: RNAse P is a ribozymeGuerrier-Takada et al, and Altman, Cell, 35, 849-857 (1983) 圣潦免净凹授果卒袜澜悬缴陕泼湿骡圾倦撕几境央琴晾钨筑罕泥恕挟叠洒-现代医药生物技术概论-现代医药生物技术概论核酶发现的意义：1.它突破了“酶是蛋白质”的传统概念。 自从1926年J.Summer首先结晶了脲素酶（Urease），并证明

15、这个结晶蛋白能催化脲素水解以来，很多酶被纯化，而且都是蛋白质。从而酶被定义为“生物催化剂是一种称作酶的特殊蛋白”，“使代谢进程加速的生物催化剂”。核酸性酶的发现无疑是对传统的酶和催化理论巨大的冲击。 宣冀遣参颈遮嫡绚斩权堤莲棱赤卤疵区舟画椭奈嫂辊骗惰各恳下糯吁撑氨-现代医药生物技术概论-现代医药生物技术概论核酶发现的意义：2.核酸性酶的发现对科学家们普遍感兴趣的生命的起源这一问题有了新的认识，对生物前化学（prebiotic chemistry）有重要贡献。 长期以来对生命起源中是先有蛋白质还是先有核酸争论不休。核酸分子含有合成蛋白质的氨基酸序列的信息，而蛋白质（酶）又是在核酸修复、转录、剪接

16、、翻译等等所有环节中必须存在的。因此谁先谁后的问题就像“鸡与鸡蛋”的问题一样令人困惑。舱背堑坞陇妻欣已蝶拟殖爪蒂敞柜颅绿贬勇批叭谅赚糯矫苇泳阁躯猖掂袍-现代医药生物技术概论-现代医药生物技术概论 长期以来，人们认为所有的生命形式在冗长的相互依赖的循环中，信息分子和功能分子是分离的。核酸是信息分子，而蛋白质是功能分子。 核酸性酶的发现给人们以启示，即RNA分子不但有复制的功能，含有复制的信息，而且还有催化的功能。因此既是信息分子，又是功能分子。这些新的概念无疑将对生物前化学的研究开辟新的思路。迫仿疾呻乐嗽哇尝搀腕蠢寒函傣加辛翌显詹盈望探蛇碟樱崭锗亩顶疮冲皿-现代医药生物技术概论-现代医药生物技术

17、概论How many ribozyme ? Why ?- the hammerhead ribozyme (plant virus)- the hairpin ribozyme (plant virus)- hepatitis delta ribozyme (human virus)- neurospora VS ribozyme (mitochondrial RNA)- group I and group II intron ribozyme (rRNA and mt RNA)- RNAse P (tRNA maturation)- Ribosome (translation)- Splic

18、eosome ? (splicing)瘟索岩臭渍诞鳃呢爱焙戎莱颊眶虫并嘛萍撞戳榨豪琶鹊叛跌说翰瘴然挡蟹-现代医药生物技术概论-现代医药生物技术概论核酶的酶活性反应种类:核苷酸转移作用磷酸二酯键水解作用 磷酸转移反应催化作用 脱磷酸作用限制性内切酶作用肽基转移作用默孰康阮编贾户戍瞻局拳几泼鞠秋歌梗星隧丫跃寓逾玉朽近逊膳腥梗要钩-现代医药生物技术概论-现代医药生物技术概论One main reaction: Nucleolytic cleavageTransesterification (SN2)From Lilley TIBS (2003)HammerheadHaipinHepatitis de

19、ltaVS ribozyme抡焦焊弓饯始辜掘孪索枚铡可呛庆蚂炽爬拒戎来呻橇堆猜泼辆谈衫翌庶庞-现代医药生物技术概论-现代医药生物技术概论The hammerhead ribozyme (plant virus)- discovered in small RNA satellites of small viruses (1986)- replication by rolling circle mechanismSecondary structure丘敢微酿荧没黔昆酣付供胯谆踏坞允施告窜违毅每腥氏灶抛谓恍绿潘纪唱-现代医药生物技术概论-现代医药生物技术概论The hammerhead ribozy

20、me (plant virus)- tertiary structureScott et al and Klug, Science 1996伐认蔗蛰集坑圈罩纱带估事镶哼淳湍饶撮邪贫遇黑砧忧您蝎警因扛朔拦拧-现代医药生物技术概论-现代医药生物技术概论enzyme, substrate, and cleavage site (A-1.1 and C-17)Hammerhead ribozymesVoet. 31-53 Small ribozymes in RNAs of certain plant viruses Reaction: transesterification. A 3-5 phosp

21、hodiester bond between two adjacent nucleotides (red) is cleaved. A 2-3 cyclic phosphodiester and a 5OH result from the clevage: 痢远瞻挝婶崩拨沥旭弹嫩碧母空驴熊靖辐堰侣肪绿囱峡修琳的庙咨提未翱-现代医药生物技术概论-现代医药生物技术概论Hairpin ribozymeNature 418, Doudna and Cech, 2002The hairpin ribozyme is a catalytic RNA derived from the self-cleavi

22、ng and ligating domain of the satellite RNA of tobacco ringspot virus. Reversible transesterification catalyzed by the ribozyme城诸倚腑筷植墩健搂晚痈宁文峙蛛驱划夸泣戮陆妨谰嗜粹广麓令渣参祈敦-现代医药生物技术概论-现代医药生物技术概论The hepatitis delta ribozyme (human virus)From Lilley TIBS (2003)更印盾廖热咀舶剖逃还企末陶凶渣屋地凳擂雇烘偿羊捂儡微捧笨蚂稼序禽-现代医药生物技术概论-现代医药生物技术概论

23、General structure of group II intronsRNA World 11.1adenine that drivesthe first nucleophilic attack(step 1).arrows point towards the exon-intron junctionsexons as dashed lines.输镣爆哀豁邦咸癸楔泛告捶祈棕捷逝妒楷粱毯柔弯映毗铺墟司柳炔源灯娃-现代医药生物技术概论-现代医药生物技术概论 Group I &II intron ribozyme (rRNA and mt RNA)Doudna and CechNature, 2

24、002钝事超酪觉恃捌久积搀仰甜咨姆拘档吱乒疟陨埠筷束缘言瞻金懈灶拒劝厄-现代医药生物技术概论-现代医药生物技术概论 Group I intron ribozyme (rRNA and mt RNA)Golden et al, and cechScience (1998)问气雪血曹姓坯阀偶理汽荧航戒谢恤葱按君炬填斥备獭燃淳卫混扑视潍浑-现代医药生物技术概论-现代医药生物技术概论Catalytic efficiency, condition- ribozyme follows a Michaelis-Menten kineticsE + S ES E + P k1k2k-1- all ribozy

25、me need cations for activity (M2+ , M+) Km= k-1+ k2 k1= 10-5-10-7 Mkcat= 0.5-2 min-1kcat/ Km= 103-106 M-1.min-1Good catalytic efficiency!轩粒轧嗽脏虫硬尝抢骋沸甸赋慎硷字偶萤嗅耻钦咯拐彰巨涨扮冉朽秀介龚-现代医药生物技术概论-现代医药生物技术概论核糖体是一个核酶Ban, et al., Science, 289, 905(2000)Peptidyl transferase center讼纷件个固折触呸汉妻灾请素画遗活季梨韭裁拢墩玄疯弹璃钢帆云愉台彬-现代医药生

26、物技术概论-现代医药生物技术概论Ribozyme technologyA specifically-designed ribozyme cleaves a specific pathogenic RNA molecule to make it inactive. For example, the viral RNA causing hepatitis C. Very promising results using cell cultures.The ribozyme is synthesized in vitro and administrated to the patient.Problem

27、s: half-life too short and low potency once in the body. 帆混呻击覆阁溃邹姥奏沾如导邀铝誓准裤四戳乙遥窘挑来疾忱渣脑寺挚买-现代医药生物技术概论-现代医药生物技术概论Reference:Reviews: Lilley TIBS (2003)De Rose Chem & Biol (2002)Ferre dAmare Biopolymer (2003)Article: Kruger et al, and Cech, Cell (1982) Guerrier-Takada et al, and Altman, Cell (1983) Scot

28、t et al Nature (1995) Science (1996) Rupert et al Nature (2001), Science (2002)蓬营珊祝擂讶矩款区鸥碧驾科腐碎坑猖糜蕾汞富先孟洒恶危洗学鸥谭拆杰-现代医药生物技术概论-现代医药生物技术概论Part II: SELEXA brief HistoryThe method ?A few examples.Biological application ?壕韭筹例伺辩梭遏霞嘴桌婉度鞍印趁织侗倒锌岭脚订哭愁玛蚁慎葡膏迅翅-现代医药生物技术概论-现代医药生物技术概论适配分子(aptamer) SELEX(指数级富集的配体系统进化技

29、术): Systematic Evolution of Ligands by EXponential enrichmentA brief HistoryEllington and Szostak, Nature (1990)Tuerk and Gold , Science (1990)歼弦听鸵逻来奥甩泵肠沤奶姆容伯贸宿逞或摘躇汲橙哟携商叮怠道跋京吕-现代医药生物技术概论-现代医药生物技术概论In vitro selection of RNA molecules that bind specific ligands Andrew D.Ellington&Jack W.Szostak Subpop

30、ulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 1010 random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.Systematic evolution of li

31、gands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Tuerk C, Gold L.High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. M

32、ultiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this proced

33、ure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNAs to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinit

34、y ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.洞拿罕伟咙字捉京角氧盲边抹钧沂妄曼斌砸哲课安痹帮嘱尔褐熏佛也徐弧-现代医药生物技术概论-现代医药生物技术概论In-Vitro Selection and In-Vitro Evolution(SELEX)AMPLICATIONSELECTIONMUTATIONInitialpool哈耽幌缝鲁摹

35、帮塞骋胞婚王钓抬舒胸罚咋例铺塘忙差蠢滚椿侠掏悍用犀靛-现代医药生物技术概论-现代医药生物技术概论Wilson and Szostak, Ann.Rev.Bioc. (1999)盔玄诱弄钟锅秽丫忻谚捎像嘉习闯寿歌谁艾吝劲脊搓坟乐粕慌达蝶饲文祖-现代医药生物技术概论-现代医药生物技术概论- Selection against small molecules- Selection against proteins- Selection of new ribozymes (RNA world)臆萄走坚员口褂脱怀斡筛筋署驻沥狱坝客策肉候莫滋荣赴粗忍帝减耶坦后-现代医药生物技术概论-现代医药生物技术概论T

36、he ATP aptamer structure蝗轻祷论千鸿投驯蛊泄钥菊筷栋刑住熔兄喻蛮滔终纺莹册越窿助菠幻椿协-现代医药生物技术概论-现代医药生物技术概论In vitro selection of an enzyme彻但篮痪蒸湃眷荆萤传漆欺黍酿瑟焉署鸣归孰镭驹掠枪撕垄枫婆艘猎紊损-现代医药生物技术概论-现代医药生物技术概论搏窝饺犀谰厅饥冬焊睹伸蝴哗恒棘秉砸昧熄崭美球渍尺娇恍航父殃狗定薪-现代医药生物技术概论-现代医药生物技术概论哄搽棚奖槛蛹钙轴柏尽岩藤几谩砚称偶枪募笛转防厉彼性关天络疤没嚼桔-现代医药生物技术概论-现代医药生物技术概论Ribozyme catalyzing peptide b

37、ond formation like ribosomeB. Zhang & T. Cech, Nature 390, 96-100 (1997)B. Zhang & T. Cech, Chemistry & Biology, 5, 539(1998) （封面）严西秀朵琉版侥放袱芳孪随叙功旭忿沃闽东诗垦刁襄垒憨步丹堵校雏芦宰-现代医药生物技术概论-现代医药生物技术概论Aptamer优点l、Aptamer的确定是通过体外过程完成的，它不依赖于细胞或动物，也不在体内，其特性可根据需要改变。2、由于不需要免疫动物和细胞，毒素和没有免疫源性的分子也可产生高亲和力的Aptamer。3、Aptamer能用化

38、学合成的方法制备，具有很高的精确性和重复性。在变性条件下能纯化得到很高的纯度。在制备Aptamer的过程中几乎没有批与批之间的差异。4、标记分子如荧光素和生物素能标记到Aptamer的精确位置，在合成过程中亦可以衍生上去。5、Aptamer能可逆变性。变性后的Aptamer能在数分钟之内复原，并能长期储存和在常温下运输。 拭珠踊躬卡赠届蜘临襄象腻喇钞搬屎日盖屋牺尹驭刀阿病硼急详曳惧珐斥-现代医药生物技术概论-现代医药生物技术概论SELEX技术和Aptamer应用SELEX技术在基础医学研究中的应用、在药物筛选中的应用、在临床诊断中的应用等。Aptamer在流式细胞分析中的应用、Aptamer在

39、传感器中的应用、Aptamer在荧光偏振分析中的应用、Aptamer在双位点分析中的应用、Aptamer与分子灯塔的结合、Aptamer在毛细管电泳中的应用、Aptamer作为分子开关、Aptamer检测固定在膜上的蛋白质、Aptamer阵列研究蛋白组等。SELEX技术的改进与发展：导向SELEX技术、复合靶分子SELEX技术、基因组SELEX技术等 淋茧窃茧鸯翌炙潜掸尝鹅质稳埔氦虚哟上缘姥隋把见衅沙谦哀袍梭氨阻捻-现代医药生物技术概论-现代医药生物技术概论Part III.反义核酸技术反义核酸（Antisense Technology）Oligonucleotide (P-S)2-Alkyl

40、 oligonucleotidePNAsMorpholinosLNA转砧歇壶烧缴仙奠券咬玲晤恕校犯朋赋懦饱窃者滤酮迅偷起账揪将稠环推-现代医药生物技术概论-现代医药生物技术概论Antisense彼纲赠握评店附匙氛稽酸骤训痒骤衡乍兰佣诫隆嘿钾偿靶慑砧塘盟吴仇琼-现代医药生物技术概论-现代医药生物技术概论Antisense technologyA single-stranded RNA or DNA molecule that is complementary to a target mRNA pairswith the mRNA and prevents translation.This str

41、ategy works well in the laboratory on cultured cells and on model organisms.Clinical example: treatment against cancers. The tumor sizes decreased but this was mainly due tothe production of interferons in response to high doses of foreign RNA. If the dose waslowered to prevent the interferon respon

42、se, the clinical benefits largely disappeared as well.呈询与塌瓜尼缆路挫蕴匆伪鲸仙螺粟捞尿往抖嗅氖梗债耙萍宅苏肃响肮米-现代医药生物技术概论-现代医药生物技术概论Antisense Mechanisms RNase-H RISC Steric dependant dependant blockingSummerton J. Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type. Biochimica et Biophysica

43、Acta 1999 1489: 141-158絮胡炎貌舌适熬绽缔励睬式嘻阀得刚拂闪吸既迸使赂琶吞事阎钠果敷可惫-现代医药生物技术概论-现代医药生物技术概论Representative Antisense StructuresRNARISCdependantMorpholinoStericblockingPhosphorothioate RNase-H dependant陋邯守前健俞颓岩李尖舔腾栏措仙辟厩忻兹税游帮祟暑缔伦戳咆寇松藐诀-现代医药生物技术概论-现代医药生物技术概论Morpholino-RNA Heteroduplex Watson-Crick bonds Bases positio

44、ned for strong binding Usually 25 base Morpholino oligos are used缀嚼韧凝弹卸殷肇羌十炙谩业琢喂霜川遗每高低满啪托霜巡刀忠峡门冒柜-现代医药生物技术概论-现代医药生物技术概论Blocking TranslationA steric-blocking oligo stops the initiation complex as it moves toward the start codon. Without reaching the start codon, the complete ribosome cannot form. Wit

45、hout the complete ribosome, the protein will not be made.落搓霸春猖跋籽糜梦兽列拄道钳扬伏关妙跟卯子喘杆鸥银印弥咙偷舍助婪-现代医药生物技术概论-现代医药生物技术概论Blocking Translation肖呵厕接把蜀匡枢欣荐毡绵缎楚浑筏凋疾脯烦嚷褒幼泅劈盂恃驾谱赫抢勾-现代医药生物技术概论-现代医药生物技术概论Blocking SplicingWhen a Morpholino binds to boundary of an exon and an intron, the Morpholino can change pre-mRNA s

46、plicing. This results in a changed mRNA and a changed protein.杰桃奈箩东钾断献瞻量灾寇七绍臆裙措晒谣喀某掠镐娄线汕喻奸骇竹茵幸-现代医药生物技术概论-现代医药生物技术概论Blocking Splicing氓媚蜒腆吓袖仇琉氛殉俺境敏半秤忿均悲躬富宙上匙是色始佰慕驶欲镊细-现代医药生物技术概论-现代医药生物技术概论FDA批准的反义核酸药物1998年Vitravene (Isis Pharmaceuticals):主治艾滋病患者中十分常见的巨细胞病毒视网膜炎。 2003年又有一个“重磅炸弹”级的反义药物Fuzeon在美国上市。此药是新一代

47、抗艾滋病病毒（HIV）新药，属于病毒的融合阻止剂类药物，能“锁定”HIV基因，使其无法发挥“融合”功能并在人类T淋巴细胞内复制，并最终消亡。2003年11月24日EN101正式批准英、以两国共同研制的EN101（Monasen）获得美国“罕见病用药新药”证书，准予在美国销售。EN101是一种直接针对产生乙酰胆碱酶的mRNA的反义类药物，而乙酰胆碱酶的过量分泌是引起重症肌无力的直接原因。2006年6月9日，美国FDA又批准了一种新型反义药物在美国上市ISIS301012（RASONS）。它是一种专门治疗家族性（遗传性）哮喘的新药，可从源头上遏制哮喘的发生，降低急性哮喘的死亡率。钒蒋匣泉皆构企出肛

48、饵吁社梨名父韦峙槛寡让恼仗抗碗伏析雾炯咆挺陷营-现代医药生物技术概论-现代医药生物技术概论FDA批准的反义核酸药物Mipomersen ：Genzyme (GENZ)与Isis Pharmaceuticals (ISIS)发表第三阶段实验数据，以mipomersen治疗同合子家族性高胆固醇血症（Homozygous Familial Hypercholesterolemia）。Mipomersen属于首个载脂蛋白B（apo-B)合成抑制剂，目前处于后期开发阶段，它可通过防止患者体内导致动脉粥样化的脂蛋白形成而降低患者体内LDL-C水平。目前，Genzyme和Isis两家公司正进行该药的一系列临

49、床实验，包括：一项治疗heFH的3期临床实验；一项治疗重度血胆脂醇过多的3期临床实验；一项治疗冠心病高危人群高胆固醇血症患者的3期临床实验；一项治疗对他汀类药物耐受性差，高危高胆固醇患者的2期临床实验。洗急票梳诅戮直景搬味抿涵阿枯险乞署税终浮鸡拯茎搅刚勃肄泅毡墩危尤-现代医药生物技术概论-现代医药生物技术概论Part IV: Introduction to RNAiA brief HistorySiRNA and miRNA RNAi MechanismA few very recent structures Biological applicationA practical example

50、of siRNA纲湿贸甘妈允占消圣蓝奶住洪龟哭够堆舞婪遥沮色桶综砍愉冗澡容唁浊表-现代医药生物技术概论-现代医药生物技术概论2006年的诺贝尔生理学奖获得者Andrew Z. FireCraig C. Mello跑喝泞粕菲吁阳憨现砍窝诵叁惰慨摈祁仟又茨伤懦稀珍捐郧柴雹骤杏异饱-现代医药生物技术概论-现代医药生物技术概论预备知识dsRNA：双链RNA，包含正义链和反义链Dicer：属于RNase 家族，是dsRNA的特异性核酸内切酶siRNA：small interfering RNA ，RNAi的关键效应分子，21-23个nt大小的双链RNARISC：RNA-inducing silencin

51、g complex，具有核酸内切、外切以及解旋酶活性RdRP：RNA-dependent RNA polymerases，是RNAi的调节因子，使RNAi可以在生物体内传递钧健及傅窿凭姿唾采寇篙毅现冬巫酿劳秧树莱柒枝煎蓖贝蒙廊唁齿规皑买-现代医药生物技术概论-现代医药生物技术概论勉匝肾债俺惨隧昆裴吃川笼森蹋肃肺躬曝醒熏豪赖骄被共咐昨纹击倚拂吕-现代医药生物技术概论-现代医药生物技术概论Cloning of par-1, a gene required for embryonic polarity Mapped par-1 to small region on genetic mapDNA re

52、arrangements associated with par-1 alleles identify a candidate gene Rescue mutant with wild-type gene? Problem: cant get large DNA clones comprising candidate geneSolution: Make antisense RNA from candidate gene and see if can phenocopy Par-1 phenotype. 柏蛔鬼福邀婚实衫瓷舷吗每桨减衫章莹歌八比直茫升艇挥黎碟野雏恭木垦-现代医药生物技术概论-现

53、代医药生物技术概论Caenorhabditis elegans摈幼绑驾柯偶咎寺粮全瞻给袱师更赞宵奏疮睬哥块乐雄后筛撕球铁军父燎-现代医药生物技术概论-现代医药生物技术概论RNA-mediated interferenceAntisense RNA par-1Sue Guos “antisense” experimentSue Guo, 1995Graduate Student in Ken Kemphues lab, 馏门慢汉反颖纤妮攻侠竭忠凯隘熄更见皆橙都擅参壳掣铃童陋抽渣袄知毛-现代医药生物技术概论-现代医药生物技术概论Sue Guos “antisense” experiment Cel

54、l 81, 1-20 1995Molecule Injected% embryonic lethalityZC22 (par-1)antisense52ZC22 (par-1)sense54TS antisense 0Z1 antisense 0H20 0“Surprisingly, injection of sense ZC22 RNA also induced par-1 phenotypes. It is not clear what accounts for this effect The basis for the sense effect is under investigatio

55、n and will not be discussed further”蚌翼贷祈有帖韧迭茶苔渐牡又色李佳寅梦乒滴而胡楷妒绚坏渴晰寇赵剿喀-现代医药生物技术概论-现代医药生物技术概论Andy Fire and Craig Mellos Experiment Nature 391, 806 1998Molecules Injected% twitchingunc-22 “pure” antisense0unc-22 “pure” sense0unc-22 sense + antisense 100“The mixture was at least two orders of magnitude m

56、ore effective than either single strand alone in producing genetic interference.The lowest dose tested, 60,000 molecules of each strand per adult, led to twitching phenotypes in an average of 100 progeny. At this point, the original injected material would be diluted to at most a few molecules per c

57、ell.”凡侈宗离榷忧险谚莱荒弄瓷酚橱钾朵糯洗毖虏任潜坡奉邪淡盯抹厕滤葡拢-现代医药生物技术概论-现代医药生物技术概论mex-3B dsRNANo injectionNo probeNo injectionProbe with mex3AInject mex3B antisenseProbe with mex-3AInject mex3B dsRNAProbe with mex-3AEffects of mex-3 RNA interference on levels of the endogenous mRNA (胚胎的原位杂交)舶抑羡王桐围涅语竞疮垄叛蒜得病涩爸雌觅北练卡核片羡郴烤匹嚷莹媚

58、彦-现代医药生物技术概论-现代医药生物技术概论RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Zamore PD, Tuschl T, Sharp PA, Bartel DP.Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Usin

59、g a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of

60、 the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the