Surynt Piotr, Wojtczak Blazej A, Chrominski Mikolaj, Panecka-Hofman Joanna, Kwapiszewska Karina, Kalwarczyk Tomasz, Kubacka Dorota, Spiewla Tomasz, Kasprzyk Renata, Holyst Robert, Kowalska Joanna, Jemielity Jacek
Division of Biophysics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093, Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland.
Org Biomol Chem. 2024 Aug 22;22(33):6763-6790. doi: 10.1039/d4ob01019a.
The trimethylguanosine (TMG) cap is a motif present at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions and, potentially, , particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'--position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (/) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.
三甲基鸟苷（TMG）帽是小核RNA 5'端存在的一种基序，小核RNA参与RNA剪接。TMG帽在RNA加工和稳定性中起关键作用，因为它保护RNA分子不被核酸外切酶降解，并促进其从细胞核输出。此外，TMG帽在snurportin（一种促进核输入的蛋白质）识别snRNA中发挥作用。TMG帽类似物在生化实验中用作分子工具来替代天然TMG帽。为了扩大可用的基于TMG的工具范围，我们在此将TMG帽与荧光分子转子（FMR）偶联，以开启检测蛋白质-配体相互作用的可能性，并有可能特别可视化与snurportin的相互作用。因此，我们报告了34种不同修饰的TMG帽-FMR偶联物的合成及其作为snurportin分子探针的评估。作为FMR，我们选择了三种绿色荧光蛋白样发色团（源自绿色荧光蛋白）和一种呫吨啶衍生物。对snurportin结合亲和力的评估意外地显示，对于在鸟苷2'-位置含有FMR的TMGpppG衍生二核苷酸具有很强的稳定作用。这些新发现的化合物是具有纳摩尔（解离常数）值的强效snurportin配体，比天然TMGpppG低两个数量级。对于相应的3'-区域异构体，这种效应降低了约50倍。为了加深对构效关系的理解，我们合成并测试了缺乏TMG帽部分的FMR偶联物。这些由分子对接支持的研究表明，增强的亲和力源于FMR部分提供的额外疏水接触。最强的snurportin配体，在与蛋白质饱和时也给出最大的荧光增强（/），在活细胞中进行测试，以通过荧光寿命监测检测相互作用并可视化复合物。这种方法在RNA加工和RNA-蛋白质相互作用的研究中具有潜在应用。
