Department of Chemistry, Faculty of Science, McGill University, Montreal, QC H3A 0B8, Canada.
Molecular Forecaster Inc. 910-2075 Robert Bourassa, Montreal, QC H3A 2L1, Canada.
Nucleic Acids Res. 2024 Apr 12;52(6):3164-3179. doi: 10.1093/nar/gkae118.
The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN) and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.
核糖开关能够结合其天然配体发生构象变化，这种能力与其功能角色密切相关，是抗微生物药物设计的一个有吸引力的目标。在这里，我们建立了一种基于探针的荧光各向异性测定法，以高灵敏度和高通量监测核糖开关的构象转变。我们使用枯草芽孢杆菌 yitJ S-Box（SAM-I）、梭状芽胞杆菌核梭形杆菌 impX RFN 元件（FMN）和霍乱弧菌核糖开关的 I 类环二鸟苷酸作为模型系统，开发了短荧光 DNA 探针，这些探针能够特异性识别无配体或配体结合的核糖开关构象状态。我们表明，天然配体浓度的增加会导致荧光各向异性的可测量和可重复的变化，这与通过天然凝胶分析观察到的核糖开关构象变化相关。此外，我们将该测定法应用于几种配体类似物，并证实它可以区分结合并触发天然构象变化的配体与不引起构象变化的配体。这个新平台为高通量筛选化合物文库以鉴定专门针对核糖开关功能构象变化的潜在新抗生素提供了可能性。
