Selection of RNA-Cleaving TNA Enzymes for Cellular Mg²⁺ Imaging

Catalytic DNA-based fluorescent sensors have enabled cellular imaging of metal ions such as Mg²⁺ . However, natural DNA is prone to nuclease-mediated degradation. Here, we report the in vitro selection of threose nucleic acid enzymes (TNAzymes) with RNA endonuclease activities. One such TNAzyme, T17-22, catalyzes a site-specific RNA cleavage reaction with a k_cat of 0.017 min^-1 and K_M of 675 nM. A fluorescent sensor based on T17-22 responds to an increasing concentration of Mg²⁺ with a limit of detection at 0.35 mM. This TNAzyme-based sensor also allows cellular imaging of Mg²⁺ . This work presents the first proof-of-concept demonstration of using a TNA catalyst in cellular metal ion imaging.