Click-iT trinucleotide cap analog: Synthesis, mRNA translation, and detection

The first example of the synthesis of a new trinucleotide cap analog containing propargyl group such as m7,3 '-O-propargylG(5 ')PPP(5 ')AmpG is reported. The effect of the propargyl group in trinucleotide analog with a standard trinucleotide cap analog (GAG), m7G(5 ')ppp(5 ')AmpG was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured A549 lung carcinoma epithelial cells. The new propargyl cap analog is a substrate for T7 RNA polymerase. Notably, the mRNA capped with the propargyl cap is translated similar to 1.3 times more efficiently than the mRNA capped with the GAG cap. The most characteristic feature of the new propargyl cap analog is that the presence of the propargyl group allows further modification of the mRNA by chemical ligation of an azide-containing fluorescent-labeled substrate to the mRNA via click chemistry

Automated summary

The synthesis of a new trinucleotide cap analog containing a propargyl group has been reported. The propargyl group in the analog was found to enhance capping efficiency, T7 RNA polymerase transcription efficiency, and translation activity. It also allows for further modification of the mRNA through chemical ligation. The propargyl cap analog showed a 1.3-fold increase in translation efficiency compared to the standard cap analog.