Enzyme-Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells.

Bioorthogonal control of metal-ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18-base pair recognition site of a homing endonuclease (I-SceI), which is found by chance only once in 7×1010  bp of genomic sequences, and can thus form a near bioorthogonal pair with I-SceI for DNAzyme activation with minimal effect on living cells. Once I-SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg2+ to release the fluorophore-labeled DNA fragment and produce a fluorescent turn-on signal for Mg2+ . Thus I-SceI bioorthogonally activates the 10-23 DNAzyme for imaging of Mg2+ in HeLa cells.