Abstract
RNA-RNA crosslinking provides a rapid means of obtaining evidence for the proximity of functional groups in structurally complex RNAs and ribonucleoproteins. Such evidence can be used to provide a physical context for interpreting structural information from other biochemical and biophysical methods and for the design of further experiments. The identification of crosslinks that accurately reflect the native conformation of the RNA of interest is strongly dependent on the position of the crosslinking agent, the conditions of the crosslinking reaction, and the method for mapping the crosslink position. Here, we provide an overview of protocols and experimental considerations for RNA-RNA crosslinking with the most commonly used long- and short-range photoaffinity reagents. Specifically, we describe the merits and strategies for random and site-specific incorporation of these reagents into RNA, the crosslinking reaction and isolation of crosslinked products, the mapping crosslinked sites, and assessment of the crosslinking data.