RAP-RNA: A Methodological Approach to Study Base-Pairing Between Endogenous RNAs.

Long non-coding RNAs (lncRNAs) engage in RNA-RNA interactions (RRIs) that shape transcript fate and gene regulation, yet these duplexes are often transient and difficult to capture in cells. This chapter presents RNA Antisense Purification-RNA coupled to psoralen crosslinking (RAP-RNA[AMT]), a targeted method to capture direct RNA-RNA base-paired partners of a chosen lncRNA in its native cellular context. Duplexes are stabilized with 4'-aminomethyltrioxsalen (AMT) and UVA; the bait lncRNA is captured with biotinylated antisense probes under high stringency, and RNA partners associated through direct base pairing are released by RNase H digestion and quantified by RT-qPCR. The protocol provides practical guidance on probe design, crosslinking parameters, essential controls, and quantitative analysis. Together, the step-by-step workflow enables high-specificity mapping of lncRNA-centered RRIs and can be adapted for focused validation across diverse targets and cell types.