Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus
Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus
复制标题
DOI：
10.1038/sj.gt.3302002
复制
发表时间：
2003-08-01
期刊：
影响因子：
5.1
通讯作者：
Guo, P
中科院分区：
文献类型：
--
作者：
Hoeprich, S;Zhou, Q;Guo, P
细菌病毒φ29的DNA包装pRNA可形成二聚体，进而形成六聚体，它包含两个独立的紧密自折叠结构域。构建的环形排列pRNA不影响pRNA的折叠。用可变序列连接pRNA的5′/3′末端也不会干扰其折叠和功能。这些独特的特性有助于防止两种常见问题——细胞中的核酸外切酶降解和错误折叠，使pRNA成为携带治疗性RNA的理想载体。设计了一种基于pRNA的载体来携带切割乙肝病毒（HBV）多聚A信号的锤头状核酶。将靶向HBV的嵌合核酶作为环形排列的pRNA连接到pRNA的5′/3′末端。使用两个顺式切割核酶位于嵌合核酶两侧并对其进行加工。包括用于靶向HBV的两个臂的锤头状核酶在pRNA的护送下能够正确折叠。嵌合核酶在体外几乎完全切割HBV mRNA的多聚A信号。细胞培养研究表明，与没有pRNA护送的核酶相比，嵌合核酶能够增强对HBV复制的抑制作用，这通过Northern杂交和e抗原检测得以证明。pRNA还可携带另一种锤头状核酶来切割其他RNA底物。这些发现表明，pRNA可作为一种载体，在体内赋予核酶、反义RNA和其他治疗性RNA分子稳定性。
The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5'/3' ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems - exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5'/3' ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.