Crielaard Stefan, Peters Casper F M, Slivkov Alexandar, van den Homberg Daphne A L, Velema Willem A
Institute of Molecules and Materials, Radboud University Heyendaalseweg 135 Nijmegen 6525 AJ The Netherlands
Chem Sci. 2025 Feb 11;16(11):4725-4731. doi: 10.1039/d4sc06189f. eCollection 2025 Mar 12.
RNA is a multifaceted biomolecule with numerous biological functions and can interact with small molecule metabolites as exemplified by riboswitches. Here, we profile the transcriptome on interactions with the metabolite Thiamine Monophosphate (TMP). We designed and synthesized a photoaffinity probe based on the scaffold of TMP and applied it to chemotranscriptomic profiling. Using next-generation RNA sequencing, several potential interactions between bacterial transcripts and the probe were identified. A remarkable interaction between the TMP probe and the well-characterized Flavin Mononucleotide (FMN) riboswitch was validated by RT-qPCR, and further verified with competition assays. Localization of the photocrosslinked nucleotides using reverse transcription and docking predictions of the probe suggested binding to the riboswitch aptamer. After examining binding of unmodified TMP to the riboswitch using SHAPE, we found selective yet moderate binding interactions, potentially mediated by the phosphate group of TMP. Lastly, TMP appeared to enhance gene expression of a reporter gene that is under riboswitch control, while the natural ligand FMN displayed an inhibitory effect, hinting at a potential biological role of TMP. This work showcases the possibility of chemotranscriptomic profiling to identify new RNA-small molecule interactions.
RNA是一种具有多种生物学功能的多面性生物分子，并且能够与小分子代谢物相互作用，核糖开关就是一个例子。在此，我们描绘了与代谢物硫胺单磷酸（TMP）相互作用的转录组。我们基于TMP的支架设计并合成了一种光亲和探针，并将其应用于化学转录组分析。使用下一代RNA测序，鉴定了细菌转录本与该探针之间的几种潜在相互作用。通过RT-qPCR验证了TMP探针与特征明确的黄素单核苷酸（FMN）核糖开关之间的显著相互作用，并通过竞争试验进一步验证。使用反转录对光交联核苷酸进行定位以及对该探针进行对接预测表明其与核糖开关适体结合。在用SHAPE检测未修饰的TMP与核糖开关的结合后，我们发现了选择性但适度的结合相互作用，可能由TMP的磷酸基团介导。最后，TMP似乎增强了受核糖开关控制的报告基因的基因表达，而天然配体FMN则显示出抑制作用，这暗示了TMP的潜在生物学作用。这项工作展示了化学转录组分析用于识别新的RNA-小分子相互作用的可能性。
