Metal-Dependent DNAzymes for the Quantitative Detection of Metal Ions in Living Cells: Recent Progress, Current Challenges, and Latest Results on FRET Ratiometric Sensors

Many different metal ions are involved in various biological functions including metallomics and trafficking, and yet there are currently effective sensors for only a few metal ions, despite the first report of metal sensors for calcium more than 40 years ago. To expand upon the number of metal ions that can be probed in biological systems, we and other laboratories employ the in vitro selection method to obtain metal-specific DNAzymes with high specificity for a metal ion and then convert these DNAzymes into fluorescent sensors for these metal ions using a catalytic beacon approach. In this Forum Article, we summarize recent progress made in developing these DNAzyme sensors to probe metal ions in living cells and in vivo, including several challenges that we were able to overcome for this application, such as DNAzyme delivery, spatiotemporal control, and signal amplification. Furthermore, we have identified a key remaining challenge for the quantitative detection of metal ions in living cells and present a new design and the results of a Förster resonance energy transfer (FRET)-based DNAzyme sensor for the ratiometric quantification of Zn²⁺ in HeLa cells. By converting existing DNAzyme sensors into a ratiometric readout without compromising the fundamental catalytic function of the DNAzymes, this FRET-based ratiometric DNAzyme design can readily be applied to other DNAzyme sensors as a major advance in the field to develop much more quantitative metal-ion probes for biological systems.