Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA.
Center for RNA Technologies and Therapeutics, University of California San Diego, La Jolla, CA, USA.
Nat Commun. 2024 Aug 16;15(1):7067. doi: 10.1038/s41467-024-50363-4.
RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.
RNA 结合蛋白 (RBPs) 在 RNA 代谢中具有关键作用，但目前的方法在检索内源性生物环境中的 RBP-RNA 相互作用方面存在局限性。在这里，我们开发了 INSCRIBE（原位敏感捕获生物环境中的 RNA-蛋白质相互作用），通过将纯化的 APOBEC1-纳米抗体融合物精确导向感兴趣的 RBP，原位 RNA 标记克服了这些挑战。该方法能够在广泛的固定生物样本（如 HEK293T 细胞和小鼠脑组织）中高度特异性地识别 RNA 结合位点，并在天然细胞环境中准确识别 RBFOX2（UGCAUG）和 TDP-43（UGUGUG）的典型结合基序。适用于任何具有可用一抗的 RBP，INSCRIBE 能够从超低输入（相当于~5 个细胞）中灵敏地捕获 RBP-RNA 相互作用。稳健、多功能和灵敏的 INSCRIBE 工作流程特别有利于临床样本等珍贵组织，能够在 RNA 相关疾病环境中探索真实的 RBP-RNA 相互作用。
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