Lange Margaret J, Burke Donald H
Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO, USA.
Methods Mol Biol. 2014;1103:11-29. doi: 10.1007/978-1-62703-730-3_2.
Aptamers targeted to HIV reverse transcriptase (RT) have been demonstrated to inhibit RT in biochemical assays and as in cell culture. However, methods employed to date to evaluate viral suppression utilize time-consuming serial passage of infectious HIV in aptamer-expressing stable cell lines. We have established a rapid, transfection-based assay system to effectively examine the inhibitory potential of anti-HIV RT aptamers expressed between two catalytically inactive hammerhead ribozymes. Our system can be altered and optimized for a variety of cloning schemes, and addition of sequences of interest to the cassette is simple and straightforward. When paired with methods to analyze aptamer RNA accumulation and localization in cells and as packaging into pseudotyped virions, the method has a very high level of success in predicting good inhibitors.
已证明靶向HIV逆转录酶（RT）的适体在生化分析和细胞培养中均能抑制RT。然而，迄今为止用于评估病毒抑制作用的方法是在表达适体的稳定细胞系中对感染性HIV进行耗时的连续传代。我们建立了一种基于转染的快速检测系统，以有效检测在两个无催化活性的锤头状核酶之间表达的抗HIV RT适体的抑制潜力。我们的系统可针对多种克隆方案进行改变和优化，并且向盒式结构中添加感兴趣的序列简单直接。当与分析适体RNA在细胞中的积累和定位以及包装到假型病毒颗粒中的方法相结合时，该方法在预测良好抑制剂方面具有很高的成功率。
