Comparative analysis of alkyne- and desthiobiotinylated photoaffinity probes for chemotranscriptomic profiling

Understanding small molecule-RNA interactions is a crucial part in drug development and fundamental biology. Chemotranscriptomic profiling is emerging as a powerful platform to interrogate interactions of small molecules with entire transcriptomes. This technique relies on photoaffinity probes that covalently capture small molecule RNA interactions. Most photoaffinity probes bear an alkyne handle that requires additional inefficient functionalization and purification steps after RNA capture. We sought to improve the workflow by directly desthiobiotinylating a photoaffinity probe, omitting these additional alkyne functionalization steps. Here, we compare the suitability of desthiobiotin and alkyne modified Ribocil-derived photoaffinity probes for chemotranscriptomic profiling. Our results demonstrate binding of both photoaffinity probes to their specific target, the FMN riboswitch, using in vitro transcription/translation and RT-qPCR. We also observed high unspecific interactions due to proposed weak and non-specific binding of the desthiobiotin moiety to RNA analyzed by dot blots and RT-qPCR. Finally, transcriptome-wide sequencing confirmed the unselective interaction of desthiobiotin. These findings suggest that desthiobiotin is an inefficient enrichment handle for the design of photoaffinity probes, resulting in many off-target interactions.