InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium.
Children's Cancer Institute Lowy Cancer Research, Kensington, 2033, Australia.
Sci Rep. 2019 Feb 21;9(1):2484. doi: 10.1038/s41598-019-38655-y.
Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.
转录组由几类 RNA 组成,这些 RNA 具有广泛但描述不佳的功能,其失调会导致许多疾病。因此,功能化 RNA 结合蛋白 (RBP) 的工程设计可能有许多应用。我们之前的研究表明,RanBP2 型锌指 (ZF) 结构域是研究设计单链 RBP 的合适支架。在本工作中,我们分析了 RanBP2 型 ZF 家族的各种成员的天然序列特异性,并对其与靶 RNA 的相互作用进行了表征。令人惊讶的是,我们的数据表明,具有不同 RNA 结合残基的天然 RanBP2 型 ZF 表现出相似的序列特异性,因此不能建立简单的识别码。尽管有此发现,但在家族内观察到了不同的区分能力。此外,为了靶向长的 RNA 序列并因此提高特异性,我们通过组合来自 RanBP2 型家族以及来自不同家族的 ZF 生成了一个 6-ZF 阵列,以努力获得更广泛的靶序列库。我们表明,这种嵌合蛋白在体外和活细胞中都能识别其靶序列 (20 个核苷酸)。总之,我们的结果表明,即使特异性改变的工程设计具有挑战性,ZF 在 RBP 设计中的应用仍然具有吸引力。