【作者】 孙妍红；

【导师】 张今；

【作者基本信息】 吉林大学 ， 微生物与生化制药， 2004， 硕士

【摘要】 脱氧核酶（Deoxyribozyme, DNAzyme, DNA enzyme），又称DNA酶或酶性DNA，是具有催化活性的DNA分子。脱氧核酶是继蛋白质酶、核酶之后生物催化剂家族的又一新成员。与前两者不同的是，自然界中似乎不存在天然的脱氧核酶，目前发现的脱氧核酶都是通过体外选择方法（SELEX）得到的。自Breaker等1994年首次报道了具有RNA切割DNA分子以来，人们通过体外筛选得到了多种具有催化功能的DNA分子。与核酶相比，脱氧核酶具有分子小、特异性好、催化效率高等特点，在基因治疗、疾病诊断及分子生物学分析工具等方面具有潜在应用价值。目前研究最多也最具应用价值的是10-23结构脱氧核酶，该结构由15个脱氧核苷酸组成的保守序列构成其催化活性中心，两侧各连接一段灵活的底物结合序列。从理论上只需调整其底物识别区的核苷酸组成。此脱氧核酶可切割任何含AU的RNA分子。这一特性使之在基因治疗（反义疗法）方面有巨大应用潜力，也就是意味着针对任何一种致病基因的mRNA，可以设计出具有高度特异性的脱氧核酶来抑制有害基因的表达，从而达到基因治疗的目的。本研究选用的10-23结构脱氧核酶是Santoro SW 和Joyce GF于1997年通过体外定向进化获得的一种能够切割RNA的脱氧核酶，它作为一种潜在反义基因治疗手段具有许多优势：①适用范围广，可以选择性可以切割mRNA上任意嘌呤和嘧啶的连接处。②专一性强，即使底物中只有一个碱基发生错配，也将使该酶的催化效率下降几个数量级乃至完全失效。③切割效率高，其催化效率kcat/KM可达109M-1min-1，超过所有的核酶。④与核酶相比，<WP=36>易于合成，价格便宜，结构稳定，其稳定性为核酶的100,000倍。⑤在生理[Mg2+]浓度下便可以发挥作用。⑥与反义核酸相比，脱氧核酶不仅能封闭靶mRNA，而且还可以切割靶mRNA，具有典型的酶周期特征，可以重复使用。现在一些科研人员正致力于用这一结构的脱氧核酶来进行肿瘤、病毒和一些遗传病的基因治疗,实验表明，其无论是在分子水平还是在体外细胞水平都表现出良好的性能。由于脱氧核酶是DNA，所以其无法象核酶和反义RNA那样借助于一般载体实现体内复制。脱氧核酶的外源传递极大地影响其体内传递、细胞的摄入以及其在生物体内的稳定性。为了实现脱氧核酶的体内复制，我们设计了一个新型环状结构脱氧核酶C-Dz482。这种全新结构的脱氧核酶是通过将10-23结构脱氧核酶克隆到M13mp18单链DNA载体中构建而成，其不仅成功地实现了自身体内复制，而且无论是在体外，还是在体内均表现出较高的酶活力。在体内，环状脱氧核酶被导入β-内酰胺酶产酶菌后，借助 M13mp18单链载体系统成功的实现体内复制，并表现出了较高的体内活力。总之，环状结构脱氧核酶是一种新型结构的脱氧核酶，它为脱氧核酶的体内复制提供了一条新途径，并为脱氧核酶今后的发展拓展了更为广阔的空间。更多还原

【Abstract】 Deoxyribozyme ( DNAzyme or DNA enzyme ) is a kind of molecules with catalytic activity. Following with protinase and ribozyme, deoxyribozyme is another biocatalyst. Unlike protinase or ribozyme , it seems that there is no deoxyribozyme in nature. All deoxyribozyme are found in vitro selection (SELEX) at present. Several type of DNA molecules with catalytic function derived by SELEX have been reported since Breaker found the DNA molecule with cleaving RNA activity for the first time in 1994.Compared with ribozyme deoxyriboxyme has much virtues including small molecule, well specificity ,high catalytic et. It has potential applied value in gene therapy, disease diagnose and analysis tool of molecular biology.With great applied value, 10-23 DNAzyme is studied widely. It’s conserved catalytic core contains 15 deoxynucleotides, a flexible substrate combaining-sequence on each side. Restructed the component of substrate recongnition domine, 10-23DNAzyme can cleavage any RNA molecule with AU in theory. It means that we expression of morbigenous gene. Thus the propose of gene therapy (antisense therapy ) is achieved.10-23 DNAzyme chosen in this study was discovered through in vitro selection in 1997, which cleaves any RNA targets at the purine-pyrimidine junctions. It consists of a catalytic domain of 15 nucleotides, flanked by two substrate-recognition domains of seven to ten nucleotides. As a potential candidate of antisense drug in gene therapeutics, 10-23 DNAzyme has many advantages over other <WP=38>antisense drugs. Unlike antisense oligonucleotides, 10-23 DNAzyme binds not only the target RNAs but also cleaves them. Its catalytic efficiency (kcat/KM) may reach 109 M-1·min-1, which is about 100-fold higher than that of the best ribozyme. Its stability is about 100,000-fold as that of ribozyme under physiological conditions. 10-23 DNAzyme exhibits a high flexibility for cleaving-site selectivity and substrate specificity, since it may cleave the purine-pyrimidine junctions of any RNAs and a single base mismatch in its antisense arms may decline the cleavage activity dramatically. Due to these advantages, it has usually shown satisfactory performance when used in the antisense therapy for a variety of cancers, viral diseases and others at the level of molecule, cell and even animal.10-23 DNAzyme cannot replicate in vivo using the general vectors in the same way as antisense RNAs and ribozymes and, consequently, the drug application may have to reply on the exogenous delivery. This has seriously affected the intracellular uptake, in vivo delivery and the stability of the DNAzymes. To achieve its replication, a new type of circular DNAzyme targeting at the β-lactamase mRNA was constructed through cloning the 10-23 DNAzyme into M13mp18 single-stranded vector. A circular DNAzymes, C-Dz482, was designed and characterized. The circular DNAzymes was found not only replicated in bacteria, but also exhibited high activities in inhibition of the β-lactamase activities and bacterial growth. In short, the new circular structure of DNAzyme opens an exciting possibility for the in vivo replication of DNAzyme and expands the <WP=39>space for the future development of DNAzyme.更多还原