N3BC – a powerful label for RNA, DNA and proteins alike

Functionalization of RNA is of high significance for RNA identification, sequencing and imaging. The range of reagents for functionalization includes e.g. phosphoramidite synthesis for direct incorporation of functional groups, small oligo probes for site specific functionalization and small compounds for posttranscriptional labeling. The latter is highly desirable because it can be applied to naïve RNA and even to RNA with yet unknown structure.
As recently published we developed a new multifunctional label based on a coumarin scaffold. The alkylating agent N3BC (7-azido-4-(bromomethyl)coumarin) was found to be selective for uridine in RNA and therefore any RNA can be labeled. The N3BC labeled RNA can then be applied to photo-crosslinking studies to cognate proteins or can be further derivatized by click-chemistry. The latter allows e.g. the introduction of an additional fluorophore for excitation or introduction of a biotin module.
The extent of derivatization in RNA can be easily modulated by changing conditions like N3BC concentration or DMSO content. Depending on the conditions between 1% and 50% of all uridines in RNA can be statistically labeled and it is even possible to label only uridines in loop regions. We have further applied N3BC on DNA where it is equally reactive with thymidine instead of uridine. In further experiments with proteins we were able to label streptavidin while keeping its biotin binding abilities.