Department of Regulatory Bioorganic Chemistry, SANKEN (The Institute of Scientific and Industrial Research), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
Nucleic Acids Res. 2021 Sep 7;49(15):8462-8470. doi: 10.1093/nar/gkab650.
Small-molecules interacting with particular RNAs and modulating their functions are vital tools for RNA-targeting drug discovery. Considering the substantial distribution of the internal loops involving two contiguous cytosines opposite to a single-nucleotide base (Y/CC; Y = C, U or A) within the biologically significant functional RNAs, developing small-molecule probes targeting Y/CC sites should provide profound insight into their functions and roles in biochemical processes. Herein, we report ANP77 as the small-molecule probe for sensing RNA internal loop of Y/CC motifs and molecules binding to the motifs. The Y/CC motifs interact with ANP77 via the formation of a 1:1 complex and quench the fluorescence of ANP77. The flanking sequence-dependent binding to C/CC and U/CC sites was assessed by fluorometric screening, provided the binding heat maps. The quenching phenomena of ANP77 fluorescence was confirmed with intrinsic potential drug target pre-miR-1908. Finally, the binding-dependent fluorescence quenching of ANP77 was utilized in the fluorescence indicator displacement assay to demonstrate the potential of ANP77 as an indicator by using the RNA-binding drugs, risdiplam and branaplam.
小分子与特定 RNA 相互作用并调节其功能，是 RNA 靶向药物发现的重要工具。考虑到生物功能 RNA 中涉及两个相邻胞嘧啶与单个核苷酸碱基（Y/CC；Y=C、U 或 A）相对的内部环的广泛分布，开发针对 Y/CC 位点的小分子探针，应该能深入了解它们在生化过程中的功能和作用。在此，我们报告了 ANP77 作为一种小分子探针，用于感测 Y/CC 基序的 RNA 内部环和与基序结合的分子。Y/CC 基序通过形成 1:1 复合物与 ANP77 相互作用，并猝灭 ANP77 的荧光。通过荧光筛选评估侧翼序列对 C/CC 和 U/CC 位点的依赖性结合，提供了结合热图。用内源性潜在药物靶标前 miR-1908 证实了 ANP77 荧光的猝灭现象。最后，利用 ANP77 的结合依赖性荧光猝灭，在荧光指示剂置换测定中证明了 ANP77 作为指示剂的潜力，使用了 RNA 结合药物，risdiplam 和 branaplam。
