PAIR-link: a ligation-free strategy that joins two RNAs into a single sequence-preserving chimeric cDNA

Abstract

Physical proximity between RNA molecules, and between the proteins that bind them, underlies much of post-transcriptional regulation, yet the methods that convert these relationships into sequence do so at a cost. Established RNA-RNA interaction maps techniques depend on crosslinking, fragmentation, and/or proximity ligation, so each partner survives only as a short chimeric read in which full identity and sequence are not preserved. We describe PAIR-link (Priming Annealed Isothermal RNA linking), a ligation-free and fragmentation-free strategy that joins two separately transcribed RNAs into a single contiguous cDNA while retaining the sequence of both partners. Two engineered RNAs carry complementary tails at their 3 ends. These tails anneal and prime one another, and an isothermal Bst-family reverse transcriptase extends across the junction in both directions to copy both RNAs into one chimeric cDNA that can be amplified by PCR and read directly by Sanger sequencing. We first established the chemistry in vitro using synthesized oligonucleotides. Because the reaction requires a defined 3 end, we drove the barcode RNAs from RNA polymerase III promoters, whose transcripts terminate at a defined site, rather than from polyadenylated polymerase II transcripts. We then implemented PAIR-link in human cells as a reconstituted ribonucleoprotein in which each barcode RNA is tethered to one half of a split reporter protein (split-cpHaloTag or split-TurboID), so that reconstitution of the split protein brings the two RNAs together and facilitates linking. Bead-intact pull-down preserves the complex through reverse transcription and amplification. We recovered the expected sequence-verified chimeric product for reporters directed independently to the nucleus and to the cytoplasm, showing that the method operates across distinct subcellular environments. To our knowledge, PAIR-link is the first method to fuse two separately produced RNAs into a single chimeric cDNA molecule with full sequences of RNA preserved, without crosslinking, fragmentation, or ligation. We discuss the current scope of the approach, which is limited to RNAs with defined 3 ends, and outline routes toward future native transcripts and protein-interaction mapping.