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

Area of Science:

• Bioconjugation Chemistry
• Nucleic Acid Chemistry
• Chemical Biology

Background:

• The inverse electron-demand Diels-Alder (IEDDA) reaction is valuable for biomolecular conjugation.
• Current IEDDA applications in nucleic acids are limited by the need for strained or complex dienophiles.
• A need exists for versatile and bioorthogonal labeling strategies for RNA.

Purpose of the Study:

• To develop a general and bioorthogonal strategy for RNA labeling using enzyme-installable allyl modifications.
• To identify suitable tetrazine dienes for efficient IEDDA reactions with unstrained allyl groups on RNA.
• To establish allyl-modified nucleosides as versatile handles for selective RNA functionalization.

Main Methods:

• Utilized frontier-orbital analysis to select an activated tetrazine diene (1,2,4,5-tetrazine-3,6-dicarboxylate, Tz 5).
• Employed enzyme-installable N6-allyladenosine (a⁶A) and 2'-O-allyladenosine (A_a) as dienophile handles.
• Performed IEDDA reactions under nonaqueous conditions with nucleosides and RNA oligonucleotides.
• Confirmed site-specific conjugation using enzymatic digestion.

Main Results:

• Tz 5 efficiently engages electronically unactivated allyl handles in IEDDA reactions.
• Stable cycloaddition adducts were formed with a⁶A and A_a in nucleosides and RNA.
• Labeling occurred selectively at allyl-modified sites without affecting canonical bases or phosphate linkages.
• Demonstrated successful RNA functionalization using this bioorthogonal strategy.

Conclusions:

• Expanded the scope of IEDDA chemistry to unstrained, electron-rich terminal alkenes in RNA.
• Established a⁶A and A_a as versatile, orthogonal handles for tetrazine-mediated RNA labeling.
• Provided a broadly applicable platform for selective RNA functionalization in biological systems.