Chemical Biology Laboratory, National Cancer Institute, Frederick, MD, 21702, USA.
Center for Cancer Research Collaborative Bioinformatics Resource, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Nat Commun. 2021 Oct 6;12(1):5856. doi: 10.1038/s41467-021-25973-x.
The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ riboswitch system, assessing sixteen diverse PreQ-derived probes for their ability to selectively modify the class-I PreQ riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ, which is similar in structure to guanine, interacts with human RNAs.
代谢物响应型核糖开关在调节细菌基因表达中的作用是众所周知的，这使得它们成为研究 RNA-小分子相互作用的有用系统。在这里，我们研究了 PreQ 核糖开关系统，评估了十六种不同的 PreQ 衍生探针，以评估它们选择性修饰 I 类 PreQ 核糖开关适体的能力。对于最活跃的探针（11），即 PreQ 的叠氮化物基光交联类似物，X 射线晶体学和基于凝胶的竞争测定证明了配体与适体的结合模式，功能测定表明探针保留了对全核糖开关的活性。使用 Chem-CLIP 的转录组全映射显示了细菌适体和探针之间高度选择性的相互作用。此外，还发现内源性人转录本中的少数 RNA 靶标特异性结合 11，为人类 RNA 中的候选 PreQ 适体提供了证据。这项工作表明了连接化学和结构对分子交联 RNA 的能力有明显的影响，揭示了 PreQ 适体/配体对在化学生物学应用中具有广泛的用途，并深入了解了与鸟嘌呤结构相似的 PreQ 如何与人类 RNA 相互作用。
