The inverse electron-demand Diels-Alder (IEDDA) reaction has emerged as a powerful tool for biomolecular conjugation, yet its application to nucleic acids has been largely limited to strained or synthetically demanding dienophiles. Here, we report a general and bioorthogonal strategy for RNA labeling by leveraging enzyme-installable allyl modifications as dienophile handles for tetrazine cycloaddition. Guided by frontier-orbital analysis, we identified 1,2,4,5-tetrazine-3,6-dicarboxylate (Tz 5) as an activated electron-deficient diene capable of engaging electronically unactivated allyl handles in IEDDA reactions under nonaqueous conditions. Tz 5 reacts efficiently with both N6-allyladenosine (a6A) and 2'-O-allyladenosine (Aa), forming stable cycloaddition adducts in nucleosides and within RNA oligonucleotides without perturbing canonical bases or phosphate linkages. The IEDDA labeling proceeds with high selectivity, and enzymatic digestion confirms site-specific conjugation exclusively at the allyl-modified nucleosides. These findings expand the scope of IEDDA chemistry to unstrained, electron-rich terminal alkenes in RNA and establish a6A and Aa as versatile, orthogonal handles for tetrazine-mediated labeling. This article offers a broadly applicable platform for selective RNA functionalization and provides new opportunities for probing, imaging, and manipulating RNA in complex biological environments.
Ge et al. (Wed,) studied this question.