Deocaris Custer C, Kaul Sunil C, Wadhwa Renu
National Institute of Advanced Indutrial Science & Technology (AIST), Tsukuba Scince City, Japan.
Methods Mol Biol. 2007;371:209-26. doi: 10.1007/978-1-59745-361-5_16.
Ribozymes are naturally-occurring catalytic RNAs from the viroid world and are being engineered in the laboratory to perform sequence-specific cleavage of a desired mRNA target. Since their Nobel Prize-winning discovery, there has been considerable interest in the utility of ribozymes as gene therapeutic agents to silence disease-causing genes. This technology is not perfect, but extensive efforts to improve upon natural design of ribozymes have enabled these RNA molecules to perform various tasks. In this chapter, we highlight the construction of two types of ribozymes: conventional and hybrid hammerhead ribozymes. The hybrid ribozyme described here is an improved version of the basic hammerhead motif with the following features: (a) the use of the RNA polymerase III (polIII) tRNAVal promoter to achieve a high level of transcription, (b) 5' linkage to the cloverleaf-shaped tRNAVal to enhance intracellular stability and cytoplasmic transport, and (c) a 3' end poly-(A) tail to act as a "molecular anchor" for endogenous RNA helicases endowing the ribozyme ability to disentangle higher-order structures of the target mRNA. Randomized hybrid ribozyme libraries have been used successfully for revelation of gene functions involved in metastasis, invasion, differentiation, apoptosis, endoplasmic reticulum stress and may be extended to gene functions involved in innate or induced cellular senescence of human cells.
核酶是来自类病毒界的天然催化RNA，目前正在实验室中进行改造，以对所需的mRNA靶标进行序列特异性切割。自其获得诺贝尔奖的发现以来，人们对核酶作为沉默致病基因的基因治疗剂的效用产生了浓厚兴趣。这项技术并不完美，但为改进核酶的天然设计所做的大量努力使这些RNA分子能够执行各种任务。在本章中，我们重点介绍两种核酶的构建：传统锤头状核酶和杂交锤头状核酶。这里描述的杂交核酶是基本锤头基序的改进版本，具有以下特点：（a）使用RNA聚合酶III（polIII）tRNAVal启动子以实现高水平转录，（b）与三叶草形tRNAVal的5'连接以增强细胞内稳定性和细胞质转运，以及（c）3'端聚（A）尾作为内源性RNA解旋酶的“分子锚”，赋予核酶解开靶标mRNA高阶结构的能力。随机杂交核酶文库已成功用于揭示与转移、侵袭、分化、凋亡、内质网应激相关的基因功能，并且可能扩展到与人类细胞先天或诱导性细胞衰老相关的基因功能。
