Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, QC, Canada.
Methods Mol Biol. 2021;2167:61-77. doi: 10.1007/978-1-0716-0716-9_5.
In vitro selection is an established approach to create artificial ribozymes with defined activities or to modify the properties of naturally occurring ribozymes. For the Varkud satellite ribozyme of Neurospora, an in vitro selection protocol based on its phosphodiester bond cleavage activity has not been previously reported. Here, we describe a simple protocol for cleavage-based in vitro selection that we recently used to identify variants of the Varkud satellite ribozyme able to target and cleave a non-natural stem-loop substrate derived from the HIV-1 TAR RNA. It allows quick selection of active ribozyme variants from the transcription reaction based on the size of the self-cleavage product without the need for RNA labeling. This results in a streamlined procedure that is easily adaptable to engineer ribozymes with new activities.
体外选择是一种成熟的方法，可以创造具有特定活性的人工核酶，或修饰天然核酶的性质。对于Neurospora 的 Varkud 卫星核酶，以前没有报道过基于其磷酸二酯键切割活性的体外选择方案。在这里，我们描述了一种基于切割的简单体外选择方案，我们最近使用该方案来鉴定能够靶向和切割源自 HIV-1 TAR RNA 的非天然茎环底物的 Varkud 卫星核酶变体。它允许根据自我切割产物的大小从转录反应中快速选择活性核酶变体，而无需 RNA 标记。这导致了一个简化的程序，很容易适应具有新活性的核酶工程。
