A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells

All aspects of RNA metabolism are regulated by RNA-binding proteins (RBPs) that directly associate with the RNA. Some aspects of RNA biology such as RNA abundance can be readily assessed using standard hybridization technologies. However, identification of RBPs that specifically associate with selected RNAs has been more difficult, particularly when attempting to assess this in live cells. The peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology has recently been developed to overcome this issue. The PAIR technology uses a cell membrane-penetrating peptide (CPP) to efficiently deliver into the cell its linked PNA oligomer that complements the target mRNA sequence. The PNA will then anneal to its target mRNA in the living cell, and then covalently couple to the mRNA-RBP complexes subsequent to an ultraviolet (UV) cross-linking step. The resulting PNA-RNA-RBP complex can be isolated using sense oligonucleotide magnetic beads, and the RBPs can then be identified by mass spectrometry (MS). This procedure can usually be completed within 3 d. The use of the PAIR procedure promises to provide insight into the dynamics of RNA processing, transport, degradation and translation in live cells.