Azide modified nucleotide closely resemble natural nucleotides and are the basis of chemoenzymatic mRNA labeling. Due to the small size of the azide group, 2′-Azido-2′-dATP is well accepted by T7 RNA polymerase and poly(A) polymerase and terminale deoxynucleotidyl transferase (Tdt) for enzymatic incorporation at the 3’end. Modified mRNA is ready for labeling with alkyne- or DBCO containing reaction partners. The possibility of performing strain-promoted Azide-Alkyne chemistry (SPAAC) enables the copper free click labeling of RNA at the sugar. This is highly beneficial, as labeling of mRNA with our approach is modular and not limited to enzymatic compatibility of bulky pre-labeled nucleotides. Moreover, only a very limited number of such pre-modified nucleotides, mainly dye-labeled ones, are commercially available.

LITERATURE

Bioorthogonal mRNA labeling at the poly (A) tail for imaging localization and dynamics in live zebrafish embryos, K. J. Westerich et al., 2020, Chemical Science, Vol. 11(11), p. 3089-3095.

https://doi.org/10.1039/C9SC05981D

Enzymatic Generation of Double‐Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases, M. Erguven et al., 2022, ChemBioChem, Vol. 23(24), e202200511.

https://doi.org/10.1002/cbic.202200511