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Abstract：
Recently, a class of heterobifunctional small molecules called ribonuclease targeting chimeras (RiboTACs) have been developed that selectively induce degradation of RNAs in cells. These molecules function by recruiting latent ribonuclease (RNase L), an endoribonuclease involved in the innate immune response, to targeted RNA structures. The RiboTACs must activate RNase L in proximity to the RNA, resulting in cleavage of the RNA and downstream degradation. To develop and validate a new RiboTAC, several steps must be taken. First, small molecule activators that bind to RNase L must be identified. Next, since RNase L is only catalytically active upon ligand-induced homodimerization, the capability of identified small molecules to activate RNase L must be assessed. RNase L-activating small molecules should then be coupled to validated RNA-binding small molecules to construct the active RiboTAC. This RiboTAC can finally be assessed in cells for RNase L-dependent degradation of target RNAs. This chapter will provide several methods that are helpful to develop and assess RiboTACs throughout this process, including recombinant RNase L expression, methods to assess RNase L engagement in vitro such as saturation transfer difference nuclear magnetic resonance (STD NMR), an in vitro assay to assess activation of RNase L, and cellular methods to demonstrate RNase L-dependent cleavage.
摘要：
最近,已经开发了一类称为核糖核酸酶靶向嵌合体(RiboTACs)的异双功能小分子,其选择性地诱导细胞中RNA的降解。这些分子通过招募潜在的核糖核酸酶（RNaseL）起作用，参与先天免疫反应的核糖核酸内切酶，靶向RNA结构。RiboTAC必须激活RNA附近的RNaseL，导致RNA的裂解和下游降解。为了开发和验证新的RiboTAC，必须采取几个步骤。首先,必须鉴定与RNaseL结合的小分子活化剂。接下来,由于RNaseL仅在配体诱导的同源二聚化时具有催化活性，必须评估鉴定的小分子激活RNaseL的能力。然后应将RNaseL激活小分子偶联至验证的RNA结合小分子以构建活性RiboTAC。该RiboTAC最终可以在细胞中评估靶RNA的RNA酶L依赖性降解。本章将提供几种有助于在整个过程中开发和评估RiboTACs的方法，包括重组RNaseL表达，在体外评估RNaseL参与的方法，如饱和转移差分核磁共振(STDNMR)，评估RNaseL活化的体外试验，和细胞方法来证明RNA酶L依赖性切割。
