This azide modified nucleotide closely resembles a natural nucleotide and enables the possibility of chemoenzymatic labeling to mRNA or ssDNA. Due to the small size of the azide group, 3′-Azido-2′,3′-ddATP is well accepted by T7 RNA polymerase, poly(A) polymerase or terminale deoxynucleotidyl transferase (Tdt) for enzymatic incorporation. This is highly beneficial, as labeling of mRNA or ssDNA 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. Internal and external studies successfully showed e.g. that the poly(A) polymerase adds specifically and sequence independently single azido terminator to the 3’-end of RNA.

The main advantage: Any mRNA or ssDNA can be site-specifically labeled without special requirements or altered production protocols at the 3’-end.

LITERATURE

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry, M. L. Winz et al., 2012, Nucleic Acids Res., Vol. 40, p. 1–13.

https://doi.org/10.1093/nar/gks062

Chemoenzymatic Preparation of Functional Click-Labeled Messenger RNA, S. Croce et al., 2020, ChemBioChem, Vol. 21, p. 1641-1646.

https://doi.org/10.1002/cbic.201900718

Application and design considerations for 3′-end sequencing using click-chemistry, M. K. Jensen et al., 2021, Methods in Enzymology, Vol. 655, p. 1-23.

https://doi.org/10.1016/bs.mie.2021.03.012