GSE168353
A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites
We report the optimization and application of diazirine photocrosslinking probes that enable selective covalent crosslinking to the PreQ1 RNA aptamer and any other interacting RNAs in close proximity.  By evaluating the impact of the linker structure and length on crosslinking efficiency we were able to shed important light on what factors have the largest impact on photocrosslinking efficiency in this model system.  Additionally, the best compound (compound 11) was shown to efficiently and selectively crosslink site-specifically to the PreQ1 aptamer even in the presence of competing human cellular RNAs.  Sequencing experiments were performed in human MCF-7 cell total RNA both with and without spike in of exogenous PreQ1 aptamer.  In the aptamer spike-in samples, the only RNA target that was significantly enriched after differential expression analysis with the negative control (compound 17) was the PreQ1 aptamer suggesting that this compound does in fact selectively crosslink to the desired target even in the presence of other RNAs.  Additionally, in the samples without spike-in of the aptamer we were able to identify 16 significantly enriched hit RNAs that may represent novel metabolite-RNA interactions.  Competitive RT-qPCR experiments confirmed enrichment of a handful of select genes with 11 and showed that the free PreQ1 ligand, which lacks the diazrine crosslinking moeity, is able to compete away compound 11.  This suggests that these interactions are specific. Overall design: Comparison of which RNAs are selectively crosslinked by 11 and 17 under various conditions through sequencing.