【作者】 于乐成；

【导师】 顾长海； 王宇明；

【作者基本信息】 第三军医大学 ， 内科学， 2003， 博士

【摘要】 随着现代分子生物学及相关学科的飞速发展，近年来发现多种具有特定生物催化功能的酶性DNA分子（catalytic DNA），即所谓脱氧核酶（deoxyribozyme）。脱氧核酶结构不同，生物催化功能也相异；其中，活性中心为5’-GGCTAGCTACAACGA-3’，能特异性剪切RNA分子的脱氧核酶在基因调控中的潜在应用价值尤为引人注目。与反义寡脱氧核苷酸（ASODN）相比，脱氧核酶分子既具有一定的反义抑制效果，又能通过改变RNA分子的空间构象以促其断裂；一个脱氧核酶分子可剪切多个RNA分子。与核酶相比，脱氧核酶识别的位点为5’…Y↓R…3’（Y＝A／G，R＝U／C），有更多可供选择的靶位，且性质相对稳定，结构相对简单。因此，它可能兼具核酶和ASODN的优势。应用脱氧核酶抗HIV-1、HPV-16、多种恶性肿瘤相关基因mRNA等的初步研究已显示了不同程度的效果，但应用脱氧核酶抗肝炎病毒的研究鲜见。病毒性肝炎至今仍缺乏充分有效的治疗措施。长期以来人们不断在探索各种可能的治疗新策略；脱氧核酶的发现为这种尝试提供了新的机遇。本课题通过分析丙型肝炎病毒5’-非编码区及部分C区（HCV 5’-NCR-C）的序列和5’-NCR的二级结构，设计4组3类共12种脱氧核酶，对其在体外分子水平、转基因肝癌细胞和感染有HCV的胎肝细胞水平的剪切和/或抑制活性进行系统评价，探讨其用作抗肝炎病毒基因调控或治疗的可能性。一、丙型肝炎病毒特异性脱氧核酶的设计和靶位筛选靶位的选择是脱氧核酶的设计基础，需结合多方面的因素加以考虑。HCV 5’-NCR高度保守，含有内部核糖体进入位点（IRES）等关键功能区。化学探测、核酸酶消化试验、热力学（自由能）推算及计算机模拟等均显示其二级结构可划分为Ⅰ～Ⅳ区。Ⅰ区可能有阻止病毒翻译启动的作用；Ⅱ区可稳定5’-NCR的空间结构及其与宿主核糖体等的结合，从而消除Ⅰ区的影响；Ⅲ和Ⅳ区是IRES的主要跨越区域，是5’-NCR最为重要的翻译启动和调控功能区。因此，靶位选择主要应在Ⅲ和Ⅳ区进行，适当兼顾Ⅱ区，避免位于Ⅰ区。5’-NCR在不同的临床分离株仍存在一定变异；为了保证所设计的脱氧核酶对各临床分离株具有普遍性作用，应避免选择存在碱基变异的区域作靶序列。此外，虽然符合5’…R↓Y…3’ 特征的位点均有可能是脱氧核酶的作用靶位，但5’…A↓U…3’ 或5’…G↓U…3’ 可能相对易被切开，是优先考虑的位点。通过测序确证质粒pHCV-neo中HCV 5’-NCR-C的序列后，根据5’-NCR的二级结构模型和脱氧核酶所能识别的底物序列特征，综合比较HCV 5’-NCR中各潜在靶位所<WP=12>处的功能区域和局部结构特点，确定第-232、-127、-84和+1共4个位点为研究对象，从而分别设计出3类脱氧核酶，即未经修饰的脱氧核酶（DRz）、两端各有两个碱基被硫代修饰的脱氧核酶（TDRz）和活性中心人工突变的脱氧核酶（MDRz）。二、丙型肝炎病毒特异性脱氧核酶在细胞外分子水平的剪切活性用限制性内切酶Nar I将pHCV-neo完全线性化，以WizardR DNA纯化系统回收长3133bp的HCV RNA 5’-NCR-C转录模板。以T7转录试剂盒体外转录获取HCV 5’-NCR-C，用CIP去除其5’ 端磷酸，再以T4聚核苷酸激酶和γ-32p-ATP进行5’ 端标记。产物用8%变性聚丙烯酰胺凝胶电泳（PAGE）分离纯化。各剪切反应体系分别含每种DRz、TDRz或MDRz 5μmol/L（两种TDRz联合应用时各为2.5μmol/L），放射性标记底物200nmol/L，在合适的pH及Mg2+浓度下，混合物经适当变性、复性并于37℃孵育一定时间后，进行8%变性PAGE，-70℃放射自显影，应用Gel DocTM 2000型凝胶成像分析仪分析各条带光密度值并计算剪切百分率。结果显示，各脱氧核酶定点剪切底物RNA的能力相差较大，以DRz-127、TDRz-127和DRz1、TDRz1的活性相对较高；37℃孵育90min后，剪切率分别达32.6%、30.8%和24.3%、21.5%。TDRz与对应DRz相比，剪切率无显著性差别，提示适当硫代修饰对脱氧核酶活性无明显影响。MDRz无产物条带，提示基本丧失剪切能力，表明脱氧核酶活性中心基序（motif）高度保守。TDRz-127和TDRz1的单独及联合剪切率分别从15min时的8.3%、7.4%和15.1%提升到90min时的31.1%、20.3%和42.6%，提示联合应用有一定的叠加效应。反应75～90min后剪切率难以进一步提升，可能与靶位是否得当、底物结合区长度是否优化以及底物二级结构的形成等有关。由于碱基构成不同，对底物结合区进行优化有可能在一定程度上改善活性。HCV RNA全长约9400nt，必然较单纯5’-NCR-C形成更复杂的二级结构，且细胞内环境较细胞外环境复杂得多；因此，有必要在细胞水平进一步评价脱氧核酶的活性。三、丙型肝炎病毒特异性脱氧核酶在转基因肝癌细胞株中的活性应用转基因细胞株和化学发光法可以快速、准确地评价脱氧核酶在细胞内对靶基因的作用效果。理论上，脱氧核酶在细胞内对靶RNA的总抑制活性包括反义抑制和定点剪切两个方面。通常设计一组无剪切活性但反义抑制效果相似的对照组（MDRz）进行比较以间接推证剪切效应。HCV 5’-NCR-C调控性荧光素酶转基因HepG2.9706细胞以每孔1×104/100μl接种于96孔培养板，孵育适当时间后给予不同浓度的脱氧核酶，给予方法包括Lipofectin转染5h、直接给予5h或24h。细胞培养完毕后，向?更多还原

【Abstract】 With the rapid development of modern molecular biology and correlated science, several kinds of short special DNA molecules with biocatalytic activity have been found in recent years. These catalytic DNAs are named as deoxyribozymes, and have different catalytic activity for the difference in their structures. Among the family of catalytic DNAs, those with the highly-conserved motif of 5’-GGCTAGCTACAACGA-3’, which acts as the active center and contributes to the ability of cleaving target RNA site-specifically, have caught the great attention of scientists for their potential usage as the tools of gene modulation or therapy. Compared with antisense oligodeoxynucleotides (ASODN), these deoxyribozymes may not only have antisense inhibition effect on target RNAs, but also own the ability to break special RNAs by distorting their configurations, and one deoxyribozyme molecule can cleave several substrate RNAs. In comparison with ribozymes, the deoxyribozymes can recognize all the sequences characterized with 5’…Y↓R…3’ (Y=A/G, R=U/C), thus have more target sites to be selected. Furthermore, they are more stable than ribozymes and have relatively simple structures. So it is considered that the deoxyribozymes may have both advantages of ASODN and ribozymes. Now the deoxyribozymes have been proved to be effective to certain extent in some incipient researches, such as cleaving RNAs of human immunodeficiency virus-1, human papilloma virus-16, and mRNAs of mutant genes in malignant tumors; but articles on their ability to cleave hepatitis virus are rarely seen.As is well known, the lack of ideally effective antiviral drugs is still an major problem in the treatment of viral hepatitis, and medical scientists have been trying their best to explore every new possible strategy to eradicate hepatitis virus in patients. The find of deoxyribozymes may bring a new hope for this purpose. In this study, we designed four groups of specific deoxyribozymes targeting at 5’-noncoding region (5’-NCR) of hepatitis C virus (HCV) according to its sequence and secondary structure, and investigated their possibility for using as antiviral measures by systematically evaluating the activity of cleavage in vitro at molecular level, inhibition or/and cleavage effect in HCV 5’-NCR-C transgeneic hepatoma cells and HCV-infected fetal hepatocytes.<WP=6>1. Selection of target sites and design of specific deoxyribozymesSelection of target sites is the essential steps for design of deoxyribozymes, and should be done according to several factors. HCV 5’-NCR is highly conserved, and containing important functional domains, such as internal ribosome entry sites (IRES). Chemical probing, nuclease digestion test, thermadynamics (free energy) calculation and computer simulation all demonstrate that the secondary structure of HCV 5’-NCR can be divided into domainⅠ～Ⅳ. Domain Ⅰ might have the ability to impede the start of HCV translation, but domain Ⅱ could counteract this effect by stabilizing the configuration of 5’-NCR and its binding to host ribosomes. Domain Ⅲ and Ⅳ are the main spanning regions of IRES, thus act as the most important functional regions in 5’-NCR for controlling initiation and modulation of HCV translation. Based on these findings, it is wise to locate the target sites in domain Ⅲ and Ⅳ preferably, or domain Ⅱ to some extent, but avoid domain Ⅰ. 5’-NCR may have some mutant bases among different clinical HCV separate strains, and sequences containing those mutant bases should not be selected as target sites in order to keep the designed deoxyribozymes have universal cleavage or/and inhibition effect on every HCV variants. On the other hand, though all sequences characterized with 5’…R↓Y…3’ might be the potential target sites, the 5’…A↓U…3’ or 5’…G↓U…3’ had been proved by Santoro et al to be more easily cleaved than other sites, thus should be selected preferably.After confirmation of sequence of HCV 5’-NCR-C from pHCV-neo in a DNA sequencer, the secondary structure of 5’-NCR was figured and all the possib更多还原