发夹核酶的研究与应用

核酶 (ｒｉｂｏｚｙｍｅ)是既能特异识别又能特异切割小分子ＲＮＡ的核酸内切酶 ,其本身也是ＲＮＡ ,主要包括发夹核酶、锤头核酶、丁肝病毒、链孢霉属ＶＳ和铅依赖性ＲＮＡ ,共同特点是可逆地切割底物ＲＮＡ的磷酸二酯键 ,生成 5′ ＯＨ和 2′ ,3′ 环磷酸末端。虽然催化产物相似 ,但它们的结构和催化机制却是很不相同的。发夹核酶 (ｈａｉｒｐｉｎｒｉｂｏｚｙｍｅ)发现于三种不同植物ＲＮＡ病毒 ,即烟草环点病毒 (ｔｏｂａｃ ｃｏｒｉｎｇｓｐｏｔｖｉｒｕｓ) ,菊苣黄色斑点病毒型 (ｃｈｉｃｏ ｒｙｙｅｌｌｏｗｍｏｔｔｌｅｖｉｒｕｓｔ…     

核酶的抗病毒作用

反义核酸技术（ａｎｔｉｓｅｎｓｅｎｕｃｌｅｉｃａｃｉｄｓｔｅｃｈｎｏｌｏｇｙ）是根据核酸杂交原理设计的以选择性地抑制特定基因表达为目的的一类核酸研究新技术,它包括反义ＲＮＡ（ａｎｔｉｓｅｎｓｅＲＮＡ,ａｓＲＮＡ）、反义ＤＮＡ（ａｎｔｉｓｅｎｓｅＤＮＡ,ａｓＤＮＡ）及核酸（ｒｉｂｏｚｙｍｅ,Ｒｚ）三大技术领域,反义核酸技术同基因治疗一样已经成为一项引人瞩目研究领域。本文对核酸作用的原理和在抗病毒方面的研究进展作一综述     

用ribozyme抑制增殖细胞核抗原的表达对HeLa细胞增殖的影响

增殖细胞核抗的（ＰＣＮＡ）是ＤＮＡ聚合酶δ的辅助蛋白,它是细胞染色体ＤＮＡ复制所必需的。人工设计的ｒｉｂｏｚｙｍｅ具有可特异地切割ＰＣＮＡｍＲＮＡ的性质,将此ｒｉｂｏｚｙｍｅ的自剪体内表达质粒导入ＨｅＬａ细胞,从细胞决ＲＮＡ中分离相应部分能在体外切割靶ＲＮＡ片段,证明此表达质粒在细胞内能表达出有活性的ｒｉｂｏｚｙｍｅ分子。与对照相比,导入ｒｉｂｏｚｙｍｅ表达质粒的ＨｅＬａ细胞进入Ｓ期的时间从１２ｈ     

丁型肝炎病毒核酶及其应用

丁型肝炎病毒（ＨＤＶ）核酶是ＤＨＶ前体ＲＮＡ分子中一段能自我切割的ＲＮＡ序列,具有独特的二级结构,为ＨＤＶ双滚环复制所必需,通过抑制其活性可以控制ＨＤＶ感染。它又存在分子间切割活性,即反式切割作用,因而有可能应用于切割异源ＲＮＡ分子。本文就这些方面的研究进展作一综述。     

目的Ribozyme两侧长片段附加序列（LAS）的去除及其转录载体构建

选择小鼠腺苷脱氨酶ｍＲＮＡ特异Ｒｉｂｏｚｙｍｅ做为目的Ｒｉｂｏｚｙｍｅ（Ｒｚ）,在目的Ｒｉｂｏｚｙｍｅ两侧分别设计５＇－顺式Ｒｚ和３＇－顺式Ｒｚ,在目的Ｒｉｂｏｚｙｍｅ上设计ＢａｍＨＩ酶切位点,并构建了上述Ｒｉｂｏｚｙｍｅ转录载体ｐＳＣＲｚ２６２,采用该质粒进行体外转录,并对转录靶ＲＮＡ分子进行了切割反应。结果显示ｐＳＣＲｚ２６２在转录过程中发生了自身切割,并释放出目的Ｒｉｂｏｚｙｍｅ－Ｒｚ２６２,该目的Ｒｉｂｏｚｙｍｅ不但去除了两侧长片段附加序列,而且保持了正确的生物学活性,通过ＢａｍＨＩ切点的设计简化了该质粒的筛选。     

Ribozyme阻断体内基因表达的研究

利用ｒｉｌｏｚｙｍｅ专一水解ＲＮＡ的特性,设计ｒｉｂｏｚｙｍｅ定点切割基因表达的中间物ｍＲＮＡ以及病毒ＲＮＡ,可以阻断基因表达和抑制病毒复制,从而达到治病和防病的目的。自１９９０年以来,利用ｒｉｂｏｚｙｍｅ技术,已经对爱滋病、肿瘤等疾病进行了大量的研究。本着重介绍ｒｉｂｏｚｙｍｅ体内阻断基因表达的应用和一些规律性问题。     

目的Ribozyme两侧长片段附加列（LAS）的去除及其转录载体构建

选择小鼠腺苷脱氨酶ｍＲＮＡ特异Ｒｉｂｏｚｙｍｅ做为目的Ｒｉｂｏｚｙｍｅ（Ｒｚ）,在目的Ｒｉｂｏｚｙｍｅ两侧分别设计５－顺式Ｒｚ和３－顺式Ｒｚ,在目的Ｒｉｂｏｚｙｍｅ上设计ＢｍＨＩ酶切位点,并构建了上述Ｒ８ｂｏｚｙｍｅ转录载体ｐＳＣＲｚ２６２,采用该质粒进行体外转录,并对转录靶ＲＮＡ分了进行了切割反应,结果显示ｐＳＣＲｚ２６２在转录过程哪生了自身切割,并释放出目的Ｒｉｂｏｚｙｍｅ－Ｒｚ２６２,该目的     

抗水稻条纹叶枯病毒核酶的设计,克隆及体外活性测定

为探索控制水稻条纹叶枯病毒（Ｒｉｃｅｓｔｒｉｐｅｖｉｒｕｓ,ＲＳＶ）设计合成了特异切割该病毒ＲＮＡ保守区及编码病害特异性蛋白（ＤｉｓｅａｓｅＳｐｅｃｉｆｉｃＰｒｏｔｅｉｎ,ＤＳＰ）基因的核酶,核酶基因的长度均为４０个碱基,用化学合成方法合成其正链及与其３＇－末端互补的１５个碱基引物,用ＴａｇＤＮＡ多聚酶合成其互补链。双链ＤＮＡ直接插入克隆载体ＰＧＥＭ３ｚｆ（＋）的Ｓｍａｌ位点。序列测定表明,克隆得到的核酶序列与设计的核酶序列完全一致。经ＳＰ６ＲＮＡ多聚酶体外转录得到核酶ＲＮＡ。当核酶ＲＮＡ与以同样方法转录得到的靶基因ＲＮＡ混合反应,可得到预期结果相同的切割片段,表明两种核酶在体外均具有特异性切割活性。     

发卡式核酶结构与功能的研究进展

发卡式核酶的一级结构为ＲＮＡ分子,切割活性所需的最小长度为５０个核苷酸,其立体结构由四个螺旋区及数个环状区域组成,核酶结构中有１５个核苷酸是必需的,它们的改变对核酶活性和立体结构都会产生显的影响,本综述了发卡式核酶结构与功能的研究进展,并讨论了其作用机理和应用原则。     

一个双价锤头型核酶在体外对两种不同植物病毒RNA的专一切割作用

根据锤头核酶模型,设计合成了一个以黄瓜花叶病毒（ＣＭＶ）外壳蛋白（ＣＰ）亚基因组ＲＮＡ为底物的锤头型核酶（ＲＺＣ）,在证明它能有效切割该底物后,再将这个核酶与一个能专一性切割烟草花叶病毒（ＴＭＶ）移动蛋白（ＭＰ）亚基因组ＲＮＡ的锤头型核酶（ＲＺ１）相互串联构成了一个双价核酶（ＲＺ１Ｃ）,体外结果表明,这个双价核酶能与相应的单价核酶ＲＺ１和ＲＺＣ一样专一而有效地切割ＣＭＶ ＣＰ和ＴＭＶ ＭＰ ＲＮＡ     

双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用

为探讨双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用,观察双位点核酶对单一核酶体外剪切的增强作用,同时比较串联核酶和混合核酶的体外切割作用,构建了核酶Rz1,Rz3, Rz1和Rz3的串联核酶(Rz13)体外转录载体, 经体外转录后切割靶RNA. 结果表明:双位点核酶,无论是串联或混合核酶均可增强单一核酶的体外切割作用, 串联和混合核酶中的单一核酶可独立发挥作用;当串联和混合数目为2个时,两者的切割效率差别不大(P＞0.05).     

小鼠腺苷脱氨酶mRNA特异Ribozyme计算机设计

小鼠腺苷脱氨酶mRNA经计算机分析,包括:ribozyme切点位置选择,二级结构预测,基因生物学功能和基因同源性分析,筛选出四个锤头结构ribozyme.结果表明上述ribozyme底物切点两翼碱基形成发夹结构,切点位于单链环区,切点所在基因片段位于该基因生物功能区内,并同已知小鼠其它基因不同源.     

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.     

In vitro catalytic activities of DNA/RNA chimeric hammerhead ribozymes against AML1-MTG8 mRNA, a fused gene transcript in acute myeloid leukemia with t(8;21)

In order to design the best construct for therapeutic hammerhead ribozymes against AML1-MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia, we synthesized DNA/RNA chimeric ribozymes directed to the area adjacent to the fusion point between AML1 and MTG8. Catalytic efficiency and fusion gene specificity of ribozymes were examined by kinetic studies of the cleavage reactions of AML1-MTG8, AML1, and MTG8 RNAs transcribed in vitro. Ribozyme 2 (Rz2) specifically cleaved AML1-MTG8 RNA at three nucleotides downstream of the fusion junction with high efficiency. The highest cleavage efficiency was achieved by Rz4.3, which targeted non-contiguous sequences and cleaved at 19 nucleotides downstream of the fusion junction. Rz4.3 also cleaved MTG8 RNA but the cleavage efficiency was three orders of magnitude lower than that for AML1-MTG8 RNA. Therefore, Rz4.3 and Rz2 are the proper ribozymes for in vivo application to modulate gene expression of the AML1-MTG8.     

Strategies for the suppression of peroxidase gene expression in tobacco. I. Designing efficient ribozymes

Five short hammerhead ribozymes (Rzs) were constructed and tested, using a range ofin vitro reaction conditions, for catalytic activity against the mRNA encoding the lignin-forming peroxidase (TPX) of tobacco. Although all 5 Rzs were shown to be able to cleave the RNA substrate, percentage cleavage varied with pre-denaturation of Rz and substrate, incubation temperature, length of incubation and ribozyme (Rz)-to-substrate ratio. One Rz with two catalytic units and 60 nucleotides of complementary sequence in 3 regions was shown to most efficiently cleave the substrate under allin vitro conditions tested. This ribozyme cleaved better than the two single ribozymes from which it was made. The superior cleaving ability of this Rz was shown to be due to the accessibility of the chosen target site and to the increased length of the hybridizing arms spanning this accessible region of the RNA.     

Ribozyme cleavage leads to decreased expression of fibroblast growth factor receptor 3 in human multiple myeloma cells, which is associated with apoptosis and downregulation of vascular endothelial growth factor

The aim of this study was to investigate the fibroblast growth factor receptor 3 (FGFR3) mRNA cleavage by ribozymes targeting FGFR3, effect of growth inhibition and associated with mechanism on multiple myeloma (MM). We designated two ribozyme-expressing plasmids that target the FGFR3 genes, Rz52 and Rz32. In vitro catalytic activity of Rz52 and Rz32 in KMS11 cells decreased FGFR3 mRNA expression to 45% (p < 0.05) and 80% (p < 0.5), respectively, of that of the control. In vivo examination of the Rz52-transfected KMS11 clone showed that FGFR3 mRNA expression decreased to 20% (p < 0.05) of the control. In the Rz52-transfected H929 clone, FGFR3 mRNA decreased to 50% of the control. Protein expression of FGFR3 decreased to 70% of the parental KMS11 and H929 clones. DNA synthesis in the Rz52-transfected KMS11 clone decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p < 0.01) of that of the control. Ribozyme cleavage-associated increase in apoptosis of Rz52 KMS11 transfectants was twice that of the control. The inhibition of FGFR3 expression by ribozymes was associated with decreased vascular endothelial growth factor (VEGF) expression and upregulation of Flt-1 but not of the KDR receptor. Our data indicate that FGFR3 is an important cell survival and antiapoptotic factor for MM cells and that ribozyme-targeted downregulation of FGFR3 might be useful as a novel therapeutic intervention in MM characterized by t(4;14).