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Summary: Conventional proximity ligation assay (PLA) suffers from target specificity issues that curtail their accuracy on interpreting proximal interactions in cell biology. Here, we present a reliable and sensitive approach by including a fluorochrome-labeled mRNA fragment along with biotin-labeled RNA probe and a target-specific antibody, which were used to generate proximal ligation signals through linear connectors in intact cells. This protocol will be particularly useful for studying the proximal interactions between RNA binding proteins (RBPs) and their target mRNAs in cells.For complete details on the use and execution of this protocol, please refer to George et al. (2021).