抗HPV11／E2基因Ribozyme对靶RNA的体外剪切研究

为了寻找HPV11型引起的生死系统感染的治疗途径和探讨HPV的致病机理,本实验以HPV11病毒质粒为模板,扩增出HPV11型E2区644bp片段,采用pEGM-T-Easy Vector为载体,构建pTV-644克隆载体,经筛选得到克隆株,取质粒测序鉴定,采用上海生化所陈农安教授编制的锤头状Ribozyme设计软件进行计算机分析,选择Ribozyme对靶基因的最佳剪切位点,及进行基因同源性分析和生物学功能分析,选择出针对HPV11 E2靶基因的RZ2777,在最适条件下进行体外剪切反应,发现人工合成和体外转录得到的Ribozyme分子均能在相应位点准确切割靶RNA分子,选择合适的反应条件切割效率达60％以上,Km和Kcat值分别为0.63μmol／L、0.12μmol／L、RibozymeL两端的5′c－is－ribozyme和3′－cis－ribozyme自我剪切释放并未影响切割活性,但靶RNA侧翼序列影响了Ribozyme的剪切活性。实验研究表明,Ribozyme可能成为治疗HPV11型引起的尖锐湿疣的有效手段,并有望在分子水平上开辟出基因治疗HPV11病毒感染的另一新天地。     

抗HPV11／E2基因Ribozyme对靶RNA的体外剪切研究

为了寻找HPV11型引起的生殖系统感染的治疗途径和探讨HPV的致病机理,本实验以HPV11病毒质粒为模板,扩增出HPVll型E2区644bp片段,采用pGEM-T-Easy Vector为载体,构建pTV-644克隆载体,经筛选得到克隆株,提取质粒测序鉴定。采用上海生化所陈农安教授编制的锤头状Ribozyme设计软件进行计算机分析,选择Ribozyme对靶基因的最佳剪切位点,及进行基因同源性分析和生物学功能分析,选择出针对HPVllE2靶基因的RZ2777,在最适条件下进行体外剪切反应,发现人工合成和体外转录得到的Ribozyme分子均能在相应位点准确切割靶RNA分子,选择合适的反应条件切割效率达到60％以上,Km和Kcat值分别为0.63μmol/L、0.12μmol/L,RibozymeL两端的5′-cis-ribozyme和3′-cis-ribozyme自我剪切释放并未影响切割活性,但靶RNA侧翼序列影响了Ribozyme的剪切活性。实验研究表明,Ribozyme可能成为治疗HPVll型引起的尖锐湿疣的有效手段,并有望在分子水平上开辟出基因治疗HPVll病毒感染的另一新天地。     

应用核酶的RNA修复功能治疗遗传疾病

十多年前 ,人们即已利用核酶 (ｒｉ ｂｏｚｙｍｅ)定点切割ＲＮＡ分子的特性 ,开始设计核酶进行抗病毒、抗肿瘤和针对某些疾病的研究 ,取得了不少进展。目前 ,针对乙肝病毒 (ＨＢＶ)和丙肝病毒 (ＨＣＶ)的核酶治疗进入了预临床 ,而针对人免疫缺损病毒 (ＨＩＶ)的核酶治疗正在进行Ｉ期 /ＩＩ期临床试验。此外 ,核酶抗病毒研究的对象有 :Ａ型流感病毒、人乳头状瘤病毒 (ＨＰＶ)、人Ｔ细胞白血病病毒 (ＨＴＬＶ)、昆虫核多角体病毒 (ＮＰＶ)等。依据同样原理 ,在了解肿瘤发病的分子机制的基础上 ,可设计核酶进行抗肿瘤研究 ,如切割ＢＣＲ/ＡＢ…     

Ribozyime及其应用前景

已发现的Ribozyme有:GroupⅠ和GroupⅡ内含子、RNaseP、类病毒、拟病毒、卫星RNA和蝶螈卫星DNA2转录物。它们的催化机制已基本阐明。利用设计的Ribozyme可切割特定RNA分子,因而可抑制基因表达。据此,在医学上可用Ribozyme治疗某些疾病;在农业上可用Ribozyme创造抗病毒的动植物新品种。Ribozyme是可进行分子内催化(如自我剪接或自我切割)或起酶作用的RNA分子。可译为核糖核酸拟酶或核酸代酶。自八十年代初以来,发现的Ribozyme已有几十种。按其作用底物可分为自体催化和异体催化两类;按其催化方式又可分为切割型和剪接型两类。Ri-bozyme的发现表明RNA是唯一的一种既可携带遗传信息又具有生物催化功能的生物大分子。     

应用扫描隧道显微镜技术观察针对HBsAg基因转录物的核酶的二级结构

核酶(Ribozyme)是具有催化RNA切割反应功能的RNA,它可以特异性地切割RN-A。最近,核酶的一级结构已经被证实,其与底物结合时所形成的二级结构如锤头状(Hammerhead)、发夹型(Hairpin)结构,亦得到初步证实。因此,能够设计并人工合成核酶,通过特异性破坏RNA,阻断其功能,从而有效地阻止病毒的复制和繁殖。这是近年来继反义RNA之后发现的又一个抑制基因表达的有力工具,开辟了分子生物学的一个新领域。     

抗HPV16 E7-ribozyme在CV-1细胞中的稳定表达

构建了由RSV—LTR启动子带动并能在细胞内稳定复制的Ribozyme的自身修剪表达质粒pRSV—Rz523、Ribozyme反义对照质粒pRSV—AE7及人增殖细胞核抗原基因(PCNA)启动子带动的HPVl6 E7片段(+554～+686)的真核表达质粒pPCNA—E7。经G418抗性筛选获得了稳定表达Ribozyme的CV-1细胞克隆,其表达水平约为9．Opmol／lO6个细胞,其中活性Ribozyme的量大于50fmol／lO6个细胞,分离得到的Ribozyme可在体外特异切割E7靶RNA片段。通过共转染Ribozyme(或反义对照)和底物表达质粒并筛选出细胞克隆．研究了Ribozyme在细胞中对底物表达水平的影响。初步结果显示．Rihozyme的导人可使细胞内底物E7的RNA表达水平降低了90％(反义对照使E7 RNA表达降低20％)。上述结果提示：在CV-1细胞中表达的Ribozyme不仅在体外,同时在细胞内具有一定的生物学活性,有可能应用于逆转官颈癌细胞的恶性表型。     

大肠杆菌16S rRNA的核酶设计和初步应用

针对细菌rRNA研发抑制细菌增殖的新型抗菌素是抗生素研究领域的新课题。细菌rRNA与基因mRNA一样自然形成折叠卷曲高级结构,其结构上可以结合反义核酸的位点即靶点,靶点的阐明是设计有效反义核酸、核酶（Ribozyme）和脱氧核酶（DNAzyme）的关键。MAST方法固定16S rRNA,将其与寡核苷酸文库杂交筛选出靶点,获得了大肠杆菌16S rRNA的6个反义核酸结合靶点,并鉴定5个靶点有效,其中1个为高效。5个靶点的反义核酸能在通透性大肠杆菌菌株培养中不同程度地抑制其生长,针对高效靶点的核酶在转化大肠杆菌中表达而抑制其生长。     

核酶的抗病毒作用研究

自然界核梅（Ｒｉｂｏｚｙｍｅ）的发现已近１０年,通过对其结构功能关系的研究,能够找出不同核酶的结构功能区域及其必需基因,同时进行分子改造,获得新的、高效的切割靶序列的核酶。本文简述了核酶的种类及两种目前常用于分子间切割核酶的分子结构,及其应用于抗病毒研究的近况。     

抗植物病毒病基因工程的研究进展及评估

本文综述了抗植物病毒病基因工程的研究进馋·介绍了转弱毒株整长cDNA路线,利用病毒CP基因,卫星RNA,反义RNA,Ribozyme技术,病毒非结构蛋白基因,DI分子以及诱导性蛋白合成终止法等非常规抗病毒战略,并对各条路线的优劣作了评估。     

锤头状核酶在HepG2.2.15细胞内的抗HBV特性

乙型肝炎病毒 (HBV)C基因区的 3′端序列在HBV各亚型中是高度保守的 ,它编码的多肽链都富含精氨酸 ,同时与前基因组RNA的包装和病毒DNA的复制有关。因此 ,HBV中C基因的 3′端保守区是核酶介导的抗病毒研究的理想靶位点。针对上述位点设计了锤头状核酶RzC ,同时作为对照 ,设计了相应的突变型核酶dRzC。HepG2 .2 .15细胞系是由头尾相接的HBVayw亚型基因组转染肝癌细胞系HepG2而建立的一株能稳定分泌HBV各种抗原和完整病毒颗粒的细胞系。因而用HepG2 .2 .15细胞进行核酶的抗病毒研究更接近于疾病的治疗过程。为此 ,把体外转录的核酶RzC和dRzC以及核酶表达载体pCRzC和 pCdRzC直接转染HepG2 .2 .15细胞。初步结果表明 ,体外转录的核酶RzC对HBV复制的抑制很弱 ,这可能是由于核酶在细胞内快速被RNA酶降解造成的 ;而通过内源性传递产生的核酶RzC能够明显地抑制HBV的复制。结果说明 ,锤头状核酶RzC在HepG2 .2 .15细胞内显示抗病毒感染的能力 ,有可能作为HBV基因治疗的一种手段     

Absence of unspecific innate immune cell activation by GATA-3-specific DNAzymes

DNAzymes of the 10-23 family represent an important class of antisense molecules with implications for therapeutic treatment of diseases. These molecules are single-stranded oligodeoxynucleotides combining the high specificity of oligonucleotide base pairing with an inherent RNA-cleaving enzymatic activity. However, like other oligonucleotide-based molecules these substances might exert so-called off-target effects, which have not been investigated so far for this molecule class. Therefore, the present study investigates putative off-target effects of DNAzymes on innate immune mechanisms using GATA-3-specific DNAzymes that have recently been developed as novel therapeutic approach for the treatment of allergic diseases including allergic asthma. The conserved catalytic domain of 10-23 DNAzymes contains a CpG motif that may stimulate innate immune cells via Toll-like receptor 9 (TLR-9). Therefore, potential TLR-9-mediated as well as TLR-9 independent cell activation was investigated using TLR-9-transfected HEK293 cells, macrophage cell lines and primary innate immune cells. Furthermore, putative effects of GATA-3-specific DNAzymes on the activation of neutrophil granulocytes and degranulation of mast cells/basophils were analyzed. In summary, no innate immune cell-stimulating activities of the tested DNAzymes were observed in any of the systems. Consequently, use of GATA-3-specific DNAzymes may represent a novel and highly specific approach for the treatment of allergic diseases.     

核酸切割酶在病原微生物检测中的研究进展

DNA是遗传信息的重要载体,其空间构象折叠性质使其具有很多的功能。利用核酸切割酶(cleaving DNAzyme)识别特定单链DNA分子并能够切割其中某条单链的性质来构建传感器,将特异性识别过程转化为凝胶电泳表征、释放荧光、比色现象的信号输出,同时能很好的和扩增反应结合来实现信号放大。核酸切割酶通过体外筛选技术获得,可以与靶物质(小分子、蛋白质,甚至整个细胞)特异性结合。由于具有制备简单,易于修饰和良好稳定性等优点,核酸切割酶被用于构建生物传感器以检测病原微生物,已应用到现场检测甚至医疗中的体内检测,结合已经成熟的检测设备血糖仪、横流层析试纸条带进行微生物检测,并广泛地应用到生物传感、食品安全、医疗在内的重要领域中。综述了近年来核酸切割酶在微生物检测中的应用,讨论了核酸切割酶在微生物检测中的切割机理和产物、靶标以及表征手段,探索核酸切割酶在微生物实际检测中的意义。对该技术的发展前景及其面临的问题进行展望,以期核酸切割酶在微生物检测领域能够更好的发展。     

An Efficient Catalytic DNA that Cleaves L-RNA

Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min^-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.     

兰花抗病毒基因工程研究进展(综述)

兰花病毒病严重影响兰花产业的发展,研究和探索防治兰花病毒病的新技术、新途径已成为众多研究者普遍关注的焦点。本文综述目前抗兰花病毒研究中应用的各种抗病毒基因工程策略,包括病毒来源基因中的外壳蛋白基因和运动蛋白基因介导的抗性策略,RNA介导的抗病毒策略,植物自身的抗病毒基因介导的抗性策略,利用多基因介导的抗性策略,以及抗体基因介导的抗性策略等。最后对兰花抗病毒基因工程的发展及应用进行了展望。     

类过氧化物DNA酶在生物传感器领域的研究进展

随着对DNA酶研究的进展,DNA酶的很多优点已经超越了传统的蛋白质酶,具有过氧化物酶催化活性的DNA酶在电化学生物检测上拥有很大的潜力。我们简要阐述了DNA酶的特性和应用,描述了其基本性质,对DNA酶在生物分析领域的应用进行了展望。     

Improving Pb2+ detection using DNAzyme-based fluorescence sensors by pairing fluorescence donors with gold nanoparticles

For previously reported fluorescence Pb(2+) sensors, DNAzymes have lead to a significant increase in Pb(2+) detecting sensitivity and specificity. However, these sensors suffer from incomplete fluorescence quenching and require additional steps for annealing DNAzymes and substrates as well as for removing the uncoupled substrates. In this study, we successfully overcome these issues by immobilizing the substrate nucleic acids on gold nanoparticles through thiol linkages. The immobilization of the substrate molecules to the gold nanoparticles lead to almost-complete fluorescence quenching and fast Pb(2+) detection, without altering the Pb(2+) specificity of the DNAzymes. After optimizing the concentration of DNAzymes, reaction time and pH, we could detect Pb(2+) as low as 5 nM within 20 min without the preliminary and the post treatments. Considering the multi-color-fluorescence quenching capability of gold nanoparticles and the to-be-developed functional nucleic acids for other metal ions, this study could extend the application of DNAzymes to the detection of multiple heavy metal ions.     

DNAzymes to beta 1 and beta 3 mRNA down-regulate expression of the targeted integrins and inhibit endothelial cell capillary tube formation in fibrin and matrigel.

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.     

A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes

Determining kinetic constants is important in the field of RNA-cleaving deoxyribozymes (DNAzymes). Using todays conventional gel assays for DNAzyme assays is time-consuming and laborious. There have been previous attempts at producing new and improved assays; however these have drawbacks such as incompatibility with structured DNAzymes, enzyme or substrate modifications and increased cost. Here we present a new method for determining single-turnover kinetics of RNA-cleaving DNAzymes in real-time and in a high-throughput fashion. The assay is based on an intercalating fluorescent dye, PicoGreen, with high specificity for double-stranded DNA and heteroduplex DNA-RNA in this case formed between the DNAzyme and the target RNA. The fluorescence decreases as substrate is converted to product and is released from the enzyme. Using a Flexstation II multimode plate reader with built in liquid handling we could automate parts of the assay. This assay gives the possibility to determine single-turnover kinetics for up to 48 DNAzymes simultaneously. As the fluorescent probe is extrinsic there is no need for enzyme or substrate modifications, making this method less costly compared to other methods. The main novelty of this assay is the possibility of using full-length mRNA as the DNAzyme target.