Selective RNA Labeling Using Tetrazine-Based IEDDA Reactions With Allyl Adenosine Modifications

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 (A_a), 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 A_a 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.