Probing Local Folding Allows Robust Metal Sensing Based on a Na⁺‐Specific DNAzyme

Abstract

Fluorescent metal sensors based on DNA often rely on changes in end‐to‐end distance or local environmental near fluorophore labels. Because metal ions can also nonspecifically interact with DNA through various mechanisms, such as charge screening, base binding, and increase or decrease in duplex stability, robust and specific sensing of metal ions has been quite challenging. In this work, a side‐by‐side comparison of two signaling strategies on a Na+‐specific DNAzyme that contained a Na+‐binding aptamer was performed. The duplex regions of the DNAzyme was systematically shortened and its effect was studied by using a 2‐aminopurine (2AP)‐labeled substrate strand. Na+ binding affected the local environmental of the 2AP label and increased its fluorescence. A synergistic process of Na+ binding and forming the duplex on the 5′‐end of the enzyme strand was observed, and this end was close to the aptamer loop. Effective Na+ binding was achieved with a five base‐pair stem. The effect on the 3′‐end is more continuous, and the stem needs to form first before Na+ can bind. With an optimized substrate binding arm, a FRET‐based sensor has been designed by labeling the two ends of a cis form of the DNAzyme with two fluorophores. In this case, Na+ failed to show a distinct difference from that of Li+ or K+; thus indicating that probing changes to the local environment allows more robust sensing of metal ions.

Thioflavin T fluorescence and NMR spectroscopy suggesting a non-G-quadruplex structure for a sodium binding aptamer embedded in DNAzymes

“…Since G-quadruplex formation is dependent on salt, a sequence containing G-quadruplex would experience significant shifts in wavelengths and increases in peak magnitudes in the presence of K + compared to the absence of K + . [56] We previously measured the CD spectra of the trans-cleaving Ce13d DNAzyme and also the G-quadruplex control shown in Figure 1A and 1B, respectively [20]. The trans-cleaving Ce13d spectra were very similar to that of the cis-cleaving Ce13dA presented in Figure S5, suggesting that they had a similar overall folding.…”

“…[15,16,17,18] The identification of this Na + -aptamer proved instrumental in understanding the reason for the specificity of NaA43 and Ce13d DNAzymes for Na + , although the mechanism underlying specific Na + binding by the DNA still remains intriguing. [10,17,19,20] Our knowledge on specific Na + binding by DNA is limited and from the literature known, and a possible mechanism may rely on G4 structures. In such a case, the G4 structure would require a superior Na + -induced stabilization than K + , as the aptamer is known to show a higher affinity to Na + in comparison to K + especially at room temperature.…”