3′-Azido-ddTTP

Terminal azide bearing nucleotides closely resemble natural nucleotides and are the basis of chemoenzymatic mRNA labeling. Due to the small size of the functional groups of nucleotides they are well accepted by T7 RNA polymerase and poly(A) polymerase and teminale deoxynucleotidyl transferase (Tdt) for enzymatic incorporation. This is highly beneficial, as labeling of mRNA with our approach is really modular and not limited by 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 up to date. Internal and external studies successfully showed for example that the poly(A) polymerase adds specifically and sequence independently single azido terminator dA to the 3’-end of RNA, making the molecule ready for click labeling. The main advantage: Any mRNA can be site-specifically labeled without special requirements or altered production protocols. By click labeling at the 3’-end the half-life of the mRNA could be modulated, which might be an option for increased mRNA stability.