Nanopore sequencing data (Mapping small molecule-RNA binding sites via Chem-CLIP synergized with capillary electrophoresis and nanopore sequencing)

Herein, we describe optimized protocols to profile small molecule-RNA interactions and to define binding sites of the small molecules in RNAs using covalent chemistry. Various reactive modules appended to an RNA-binding small molecule were studied for cross-linking to the RNA target. Electrophilic modules, whether N-chloroethyl aniline or diazirine, have reactive profiles consistent with induced proximity, however probes with N-chloroethyl aniline were more reactive and more specific than those with a diazirine cross-linking moiety. The nucleotides where cross-linking occurred were elucidated by nanopore sequencing where the cross-link produces mutations in the corresponding cDNA formed by RT-PCR amplification of the cross-linked RNA. These approaches are broadly applicable to aid in the advancement of chemical probes targeting RNA, including identifying binding sites and using covalent chemistry to screen for RNA-binding molecules in a high throughput format.本文详述了利用共价化学（covalent chemistry）技术表征小分子-核糖核酸（RNA）相互作用，并精准定位小分子在RNA上结合位点的优化实验方案。本研究针对偶联于RNA结合小分子的各类反应性模块开展探究，以实现其与RNA靶标的交联反应。无论是N-氯乙基苯胺（N-chloroethyl aniline）还是双吖丙啶（diazirine），亲电模块的反应特性均与诱导邻近（induced proximity）机制相符；不过携带N-氯乙基苯胺的探针，其反应活性与特异性均优于带有双吖丙啶交联基团的探针。通过纳米孔测序（nanopore sequencing）可明确发生交联反应的核苷酸位点：交联事件会在经逆转录聚合酶链式反应（RT-PCR）扩增交联RNA所得到的对应互补脱氧核糖核酸（cDNA）中引入突变。本方法具有广泛适用性，可助力靶向RNA的化学探针开发，包括精准定位结合位点，以及利用共价化学技术以高通量方式筛选RNA结合分子。