Ribozyme probe based on molecular beacon for real time monitoring of enzymatic cleavage process

Ribozyme probe based on molecular beacon (MBR) for monitoring enzymatic cleavage process in real time is designed and studied. The approach relies on ribozyme substrates modified at the two arms, with a fluorescent moiety attached to the end of one arm and a non-fluorescent quenching moiety attached to the end of the other arm. MBR is employed to directly convert the cleavage information into fluorescence signal in real time. Compared with traditional approach, this method provides a no-radiolabeling, sensitive and effective way to research on the ribozyme activity, enzymatic dynamic process and ribozyme function during gene therapy. The activity of the ribozyme against hepatitis C virus RNA (HCV-RNA) is studled based on this assay.     

核酶的化学合成及其对烟草花叶病毒RNA片段的体外剪切

设计并用化学方法合成了针对烟草花叶病毒ＲＮＡ的核酶ＲＺ－１及ＲＺ－１小型化的核酶ＲＺ－２以及它们的底物ＲＮＡ片段Ｓｂ,研究了两种核酶对底物Ｓｂ的体外催化活性,并把核酶Ｒｚ－１与生物合成的相同组成的核酶Ｒｚ－１’进行了比较试验。     

核酶转基因载体的构建及在烟草原生质体中对TMV感染的抑制作用

设计切割烟草花叶病毒RNA的锤头型核酸Rz－2，分别以pKyLx7和pBin19为载体，构建含Rz－2基因及核酸和底物连接的RzSb－2基因的重组质粒，通过三亲交配将目的基因转入土壤农杆菌中的Ti质粒．重组Ti质粒通过PEG法转化烟草原生质体，同时对转化的原生质体接种TMVc一定时间后以半叶法检测原生质体中病毒的侵染性．结果表明转基因原生质体中TMV侵染性明显低于未转基因的对照，推测病毒在转基因的原生质体中复制受到干扰；且核酸Rz－2（正向）表达后作用最为明显，核酸连底物RzSb－2（正向）与RzSb～2（反向）之间有明显差别．     

核酶的发现及其在基因治疗中的应用

核酶是具有催化功能的结构性RNA分子,且大多数核酶具有剪切RNAs的功能,可以利用它们剪切信使RNA(mRNAs)调节基因表达,从而作为基因治疗的新手段.阐述核酶的发现历程,以及其在艾滋病、肝炎、肿瘤和生殖道系统感染等基因治疗的研究进展.最后,分析核酶在基因治疗中的技术优势及存在问题,并对核酶领域包括更多核酶晶体结构的确立、酶切机理的理解、核酶新的生物学功能的发现等研究进行展望.     

针对马铃薯卷叶病毒复制酶基因负链的突变核酶的克隆及体外转录

根据特异性切割马铃薯卷叶病毒中国分离株 ( PLRV-Ch)复制酶基因负链 RNA的锤头状核酶 ,设计、合成了编码与其相应的突变核酶的 c DNA,并克隆到质粒 p GEM-4 Z中 ,经序列分析及体外转录表明得到完整的突变核酶基因重组质粒 ,从而为突变核酶的转基因及进一步研究核酶在转基因马铃薯中表达引起的抗性及机理创造了条件     

Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.     

A conformational switch controls hepatitis delta virus ribozyme catalysis

Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.     

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.     

HER-2基因特异性核酶在MDA-MB-453细胞内的瞬时效应

目的确定人表皮生长因子受体-2(HER-2)特异性锤头状核酶(hammerhead ribozyme,RZ)对雌激素受体阴性乳腺癌细胞MDA-MB-453的影响.方法应用RT-PCR、免疫细胞化学检测RZ对MDA-MB-453细胞HER-2表达的影响;MTT法检测RZ对细胞增殖的影响.结果RZ转染入MDA-MB-453细胞中,RT-PCR检测出转染RZ的MDA-MB-453细胞中HER-2 mRNA表达量明显下降;免疫细胞化学方法检测细胞内的HER-2蛋白表达下降;MTT法检测细胞增殖活性明显受抑.结论RZ在MDA-MB-453细胞内有效抑制靶基因HER-2mRNA和蛋白的表达,同时可抑制细胞增殖,有助于进一步研究雌激素受体阴性乳腺癌细胞HER-2信号转导通路.     

基因治疗及其研究进展

基因治疗作为一种新型的疾病治疗手段 ,正在广泛用于血友病、糖尿病、癌症、心血管病、爱滋病等多种疾病的治疗领域 .基因治疗的一般步骤包括 :目的基因的转移和目的基因的表达两个方面 .其中目的基因的导入和目的基因表达的精确调控是两个关键步骤 .新发展的还有反义疗法以及应用核酶进行基因治疗等 .本文就基因治疗的基本步骤、基因治疗的对象、方式及基因治疗的现状和前景作一个简单介绍     

桥序列在构建人工核酶M1GS中的作用

目的：为检验连接的Gs序列是否会影响M1RNA高级结构而导致M1RNA活性改变,对核酶的催化机制进行探讨。方法：通过软件模拟对M1GS进行结构分析,筛选了一段独立折叠的桥序列连接M1RNA与GS,并通过体外切割实验检验有桥和无桥序列的核酶的活性。结果：有桥序列的M1GS具备体外特异切割底物的核酶活性,而无桥序列的M1GS核酶未表现体外切割活性。结论：证实桥序列对维持M1RNA高级结构和保持人工核酶M1GS的体外切割活性具有重要作用。     

Inducible Expression and Splicing of Candida Group I Ribozyme in E.coli

0　IntroductionThe discovery of group I intron, a catalytically activeRNA is a major challenge in the concept of enzyme. Thefirst example of an RNA molecule that forms a catalytic activesite for a series of precise biochemical reactions was reported20 years ago: the self splicing pre ribosomal RNA of theTetrahymena[1]. A year after, the catalytic activity wasreported for the RNA component of a ribonucleoproteinenzyme, ribonuclease P[2]. These findings le…     

Novel guanosine requirement for catalysis by the hairpin ribozyme

THERE is much interest in the development of 'designer ribozymes' to target destruction of RNAs in vitro and in vivo. Engineering of ribozymes with novel specificities requires detailed knowledge of the ribozyme-substrate interaction, and a rigorous evaluation of sequence specificity. The hairpin ribozyme catalyses an efficient and reversible site-specific cleavage reaction. We have used mutagenesis and in vitro selection strategies to show that RNA cleavage and ligation has an absolute requirement for guanosine immediately 3' to the cleavage-ligation site. This G is not required for efficient substrate binding, rather, its 2-amino group is an essential component of the active site required for catalysis.     

蜜瓜ACC氧化酶mRNA的成串锤头型核酶和反义RNA基因的合成和克隆

我们设计和合成了切割蜜瓜成熟果实ACC氧化酶mRNA的GUC^86、GUC^388、GUC^534的三重锤头型核酶（HhRz)和阻断转录的反义RNA（AR） 基因,并构建了二、四、八联体HhRz和AR基因。为了在体外和体内检测HhRz是否切割ACC氧化酶mRNA,在E.cloliDE3rna-菌株中克隆了同域和跨域作用的（HhRz)n-AR和靶子ACC氧化酶mRNA表达系统。又构建了植物体组成型和诱导型（HhRz)n-AR表达载体。     

逆转录病毒载体介导的Anti-ras核酶对T24ras转化3T3细胞的抑制作用

突变ｒａｓ基因存在于多种人类肿瘤细胞中,本文将可特异性切割１２位点突变ｒａｓ（Ｔ２４ｒａｓ）之核酶Ｒ８通过逆转录病毒载体导入Ｔ２４ｒａｓ转化的ＮＩＨ３Ｔ３细胞,以期检测核酶在细胞内对其靶基因的作用．结果表明Ｒ８可特异性地切割突变ｒａｓｍＲＮＡ,导致转化细胞生物学特性如细胞形态、生长速度等在一定程度上发生逆转     

人端粒酶逆转录酶核酶抑制端粒酶活性的实验研究

为有效切割端粒酶逆转录酶mRNA为抑制端粒酶活性,设计合成了针对端粒酶逆转录酶mRNA的核酶基因htert RZ及htert-5'RZ,构建了核酶基因的体外转录和真核表达质粒,将核酶转染至肿瘤细胞中,均能抑制端酶活性,核酶heterRZ稳定转染细胞后,使细胞倍增时间长,但无明显细胞凋亡,因而,二种核酶可望成为有效的端粒酶抑制剂,用于抑制肿瘤生长。