Muttach Fabian, Mäsing Florian, Studer Armido, Rentmeister Andrea
University of Münster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Str. 2, 48149, Münster, Germany.
University of Münster, Department of Chemistry, Institute of Organic Chemistry, Corrensstr. 40, 48149, Münster, Germany.
Chemistry. 2017 May 2;23(25):5988-5993. doi: 10.1002/chem.201605663. Epub 2017 Jan 31.
Elucidation of biomolecular interactions is of utmost importance in biochemistry. Photo-cross-linking offers the possibility to precisely determine RNA-protein interactions. However, despite the inherent specificity of enzymes, approaches for site-specific introduction of photo-cross-linking moieties into nucleic acids are scarce. Methyltransferases in combination with synthetic analogues of their natural cosubstrate S-adenosyl-l-methionine (AdoMet) allow for the post-synthetic site-specific modification of biomolecules. We report on three novel AdoMet analogues bearing the most widespread photo-cross-linking moieties (aryl azide, diazirine, and benzophenone). We show that these photo-cross-linkers can be enzymatically transferred to the methyltransferase target, that is, the mRNA cap, with high efficiency. Photo-cross-linking of the resulting modified mRNAs with the cap interacting protein eIF4E was successful with aryl azide and diazirine but not benzophenone, reflecting the affinity of the modified 5' caps.
阐明生物分子间的相互作用在生物化学中至关重要。光交联为精确确定RNA-蛋白质相互作用提供了可能。然而，尽管酶具有固有的特异性，但将光交联部分位点特异性引入核酸的方法却很稀少。甲基转移酶与其天然辅酶底物S-腺苷-L-甲硫氨酸（AdoMet）的合成类似物相结合，可实现生物分子的合成后位点特异性修饰。我们报道了三种带有最广泛使用的光交联部分（芳基叠氮化物、重氮环丙烷和二苯甲酮）的新型AdoMet类似物。我们表明，这些光交联剂能够被酶高效地转移至甲基转移酶的作用靶点，即mRNA帽结构上。所得修饰mRNA与帽结合蛋白eIF4E的光交联实验中，芳基叠氮化物和重氮环丙烷成功实现了交联，而二苯甲酮则未能成功，这反映了修饰后的5'帽结构的亲和力。
