TIVA tags are created initially via solid-phase synthesis with the cell-penetrating peptide conjugated afterwards. The functional components of the tag can be summarized as following:
* Biotin: binds to streptavidin beads for tag isolation.
* Cy3 fluorophore: used to validated cleavage of photocleavable linker. If cleaved, cell will appear green upon exposure to 514 nm light.
* Cy5 fluorophore: used to validate uptake into cells. If uptake is successful, and if Cy5 is not yet cleaved from the TIVA tag, energy from a 514 nm light will be absorbed via FRET from Cy3 to Cy5, where cells that have taken up the TIVA will appear red.
* PolyU 18-mer oligonucleotide: used to bind mRNA via complementary base pairing of their polyadenylated tails. Before cleavage of photocleavable linkers, it is caged by complementary base pairing to two polyA 7-mer oligonucleotides.
* PolyA 7-mer oligonucleotides: before the cleavage of photocleavable linkers, 2 polyA 7-mer molecules conjugate to polyU oligonucleotides to cage the TIVA tag, and thus prevent it from binding mRNA molecules. After photocleavable linkers are cleaved, the melting temperature decreases from 59 °C to less than 25 °C, leading to the disassociation of the PolyA 7-mer oligonucleotides from the TIVA tag.
* Photocleavable linker: links and stabilizes Cy5 fluorophore and PolyA 7-mer oligonucleotides to the TIVA tag. It is cleaved upon photoactivation.
* Cell-penetrating peptide CPP: guides the TIVA tag through cell membranes into tissues. It is linked to the TIVA tag by a disulphide bond that is cleaved once exposed to extracellular environment.