Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

The high-throughput sequencing–RNA affinity profiling (HiTS-RAP) assay enables large-scale profiling of protein interactions with RNA libraries using a simple protocol on a high-throughput sequencer. RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing–RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E–binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E–binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.