Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

This method should be of interest to researchers who are trying to isolate RNA protein complexes and identify their composition by mass spectrometry. We believe our protocol will be especially useful for purifying complexes that exist in cells in limited amounts and tend to dissociate during long multi-step purification procedures. The main advantage of this method is that it involves only a single purification step and could be used with RNA binding sites of any length and sequence.

Demonstrating some of the steps will be Xiao-cui Yang, who is a senior research technician in our group. For binding sites significantly longer than 65 nucleotides, obtain T7 generated RNA and an adaptor PCB oligonucleotide as outlined in the text protocol. Mix 20 picomoles of the RNA with 100 picomoles of the adaptor oligonucleotide in a 1.5 milliliter tube containing 100 microliters of binding buffer.