Liang Qishan, Xiang Joy S, Yeo Gene W, Corbett Kevin D
Department of Chemistry and Biochemistry, UC San Diego, La Jolla, California 92093, USA.
Department of Cellular and Molecular Medicine, UC San Diego, La Jolla, California 92093, USA.
RNA. 2025 Jan 22;31(2):150-163. doi: 10.1261/rna.080329.124.
Targeting and manipulating endogenous RNAs in a sequence-specific manner is essential for both understanding RNA biology and developing RNA-targeting therapeutics. RNA-binding zinc fingers (ZnFs) are excellent candidates as designer proteins to expand the RNA-targeting toolbox, due to their compact size and modular sequence recognition. Currently, little is known about how the sequence of RNA-binding ZnF domains governs their binding site specificity. Here, we systematically introduced mutations at the RNA-contacting residues of a well-characterized RNA-binding ZnF protein, ZRANB2, and measured RNA binding of mutant ZnFs using a modified RNA bind-n-seq assay. We identified mutant ZnFs with an altered sequence specificity, preferring to bind a GGG motif instead of the GGU preferred by wild-type ZRANB2. Further, through a series of all-atom molecular dynamics simulations with ZRANB2 and RNA, we characterized changes in the hydrogen-bond network between the protein and RNA that underlie the observed sequence specificity changes. Our analysis of ZRANB2-RNA interactions both in vitro and in silico expands the understanding of ZnF-RNA recognition rules and serves as a foundation for eventual use of RNA-binding ZnFs for programmable RNA targeting.
以序列特异性方式靶向和操纵内源性RNA对于理解RNA生物学和开发RNA靶向疗法都至关重要。RNA结合锌指（ZnF）由于其紧凑的尺寸和模块化的序列识别能力，是扩展RNA靶向工具箱的理想设计蛋白候选者。目前，关于RNA结合ZnF结构域的序列如何决定其结合位点特异性，我们所知甚少。在此，我们系统地在一个已充分表征的RNA结合ZnF蛋白ZRANB2的RNA接触残基处引入突变，并使用改进的RNA结合测序（bind-n-seq）测定法测量突变ZnF的RNA结合情况。我们鉴定出了序列特异性发生改变的突变ZnF，它们更倾向于结合GGG基序，而不是野生型ZRANB2所偏好的GGU。此外，通过对ZRANB2和RNA进行一系列全原子分子动力学模拟，我们表征了蛋白质与RNA之间氢键网络的变化，这些变化是观察到的序列特异性变化的基础。我们对ZRANB2与RNA在体外和计算机模拟中的相互作用分析，扩展了对ZnF-RNA识别规则的理解，并为最终将RNA结合ZnF用于可编程RNA靶向奠定了基础。
