Aquino-Jarquin Guillermo, Benítez-Hess María Luisa, DiPaolo Joseph A, Alvarez-Salas Luis M
Laboratorio de Terapia Génica, Departamento de Genética y Biología Molecular, CINVESTAV, México D.F., México.
Oligonucleotides. 2008 Sep;18(3):213-24. doi: 10.1089/oli.2008.0130.
Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.
三链体核酶（RZ）构型允许反式作用的RZ在多个表达盒（多重表达）中单独发挥作用，从而相对于传统表达的RZ增加靶标切割效率。尽管发夹状RZ与锤头状RZ相比具有优势，但其更长的长度和结构特征使三链体设计变得复杂。我们提出了一种基于具有反式切割能力且工程设计简单的单个发夹状RZ的三链体表达系统。该系统在体外针对来自人乳头瘤病毒16型E6/E7 mRNA的已知靶标RNA进行了顺式和反式切割动力学分析测试。单链和多重三链体RZ构建体在切割靶标方面比串联克隆的发夹状RZ更有效，这表明单个RZ的释放增强了反式切割动力学。与标准的双RZ构建体相比，用两种不同发夹状RZ构建的多重系统具有更好的反式切割效果。此外，三链体RZ在宫颈癌细胞中进行了顺式和反式切割。使用具有多重RZ的三链体构型允许对相同或不同的RNA进行差异靶向，从而提高针对不稳定靶标的潜在应用价值。这个原型将为未来基于RZ的治疗方法和技术的开发提供基础。
