CHEMICAL MODIFICATION OF HAMMERHEAD RIBOZYMES - CATALYTIC ACTIVITY AND NUCLEASE RESISTANCE
CHEMICAL MODIFICATION OF HAMMERHEAD RIBOZYMES - CATALYTIC ACTIVITY AND NUCLEASE RESISTANCE
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DOI：
10.1074/jbc.270.43.25702
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发表时间：
1995-10-27
影响因子：
4.8
通讯作者：
USMAN, N
中科院分区：
文献类型：
--
作者：
BEIGELMAN, L;MCSWIGGEN, JA;USMAN, N
对选择性修饰的 36 聚体锤头状核酶的系统研究已鉴定出一种通用的、具有催化活性且核酸酶稳定的核酶基序，该基序包含 5 个核糖残基、29-30 个 2'-O-Me 核苷酸、U4 和 U7 位置上的 1-2 个其他 2'-修饰核苷酸以及 3'-3' 连接的核苷酸“帽”。 8 在位置U4和/或U7处引入2'-修饰的尿苷残基。从所得的一组核酶中，有几种具有几乎野生型的催化活性，并且稳定性显着提高。具体而言，与全 RNA 亲本相比，在 U4 和 U7 处含有 2'-NH2 取代或在 U4 处含有 2'-C-烯丙基取代的核酶保留了大部分催化活性。它们在包括人血清在内的多种生物液体中的血清半衰期为 5-8 小时，而全 RNA 亲本核酶的稳定性半衰期仅为 0.1 分钟。添加 3'-3' 连接的核苷酸“帽”（倒置 T）不会影响催化作用，但会在纳摩尔浓度下将这两种核酶的血清半衰期延长至 >260 小时。这表明与全 RNA 亲本核酶相比，稳定性/活性总体提高了 53,000-80,000 倍。
A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2'-O-Me nucleotides, 1-2 other 2'-modified nucleotides at positions U4 and U7, and a 3'-3'-linked nucleotide ''cap.'' Eight 2'-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 substitutions at U4 and U7, or 2'-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only similar to 0.1 min. The addition of a 3'-3'-linked nucleotide ''cap'' (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.