There is currently a tremendous interest in developing bioorthogonal "click chemistry" methods for the modification of biopolymers. Very recently, inverse-electron-demand Diels-Alder reactions have received attention, but to date they have not been applied to nucleic acids. Here we describe the first example of DNA modification by inverse-electron-demand Diels-Alder reaction. We synthesized four different building blocks for 3'-terminal, 5'-terminal, and internal incorporation of norbornene dienophiles into oligonucleotides. These DNA strands were either directly reacted with suitably derivatized tetrazine dienes or first subjected to enzymatic manipulations. We demonstrate that the inverse-electron-demand Diels-Alder reaction allows efficient site-specific post-synthetic conjugation, often at a 1:1 stoichiometry, without any side reaction. The reaction works in aqueous media at room temperature, and no transition metals are required. Both short chemically synthesized oligonucleotides and long enzymatically amplified DNA strands were successfully conjugated.

Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.

We report the first example of RNA labeling based on inverse electron-demand Diels-Alder reactions. Both chemically synthesized and enzymatically transcribed RNAs were successfully modified with biotin or a fluorescent label. This approach works efficiently under mild conditions in water and does not require transition metals.

Norbornene-modified nucleoside triphosphates were synthesised and enzymatically incorporated into DNA via primer extension and polymerase chain reaction. DNA strands containing several hundred norbornene moieties were prepared and successfully labelled utilising the inverse-electron-demand Diels-Alder reaction.