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The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions

机译：二叠氮基修饰的尿苷类似物的合成及其在研究RNA-蛋白质相互作用中的应用

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The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U-Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U-Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate (UTP)-T-Dz was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. (UTP)-T-Dz was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.
机译：许多ncRNA所起的作用仍然未知。类似地，对涉及加工ncRNA的RNA结合蛋白的了解相对较少。鉴定新的RNA / RNA结合蛋白（RBP）相互作用可为更好地理解基因表达和ncRNA生物发生期间细胞内发生的复杂事件铺平道路。开发用于分离RBP的化学工具至关重要。在这种情况下，我们报道了在核碱基上带有重氮基部分的尿苷亚磷酰胺U-Dz的合成。含有U-Dz单元的RNA探针在单链DNA结合蛋白（SSB）的存在下被辐照，该蛋白也已知与ssRNA结合，并显示出有效的（15％产率）并选择性交联到该蛋白上。合成了相应的重氮嗪修饰的尿苷三磷酸（UTP）-T-Dz，并在转录测定中测试了其充当T7 RNA聚合酶底物的能力。 （UTP）-T-Dz被接受时，包含单次掺入的26mer RNA的最大产率为38％，而连续三次掺入的28mer产率为28％。因此，该尿苷类似物代表了一种方便的生化工具，用于鉴定RNA结合蛋白并阐明ncRNA发挥的作用和功能。

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