Sensitivity of a classic DNAzyme for Pb²⁺ modulated by cations, anions and buffers

GR5 is the first reported DNAzyme, which has RNA cleavage activity in the presence of Pb2+. Due to its excellent selectivity, GR5 has been a popular DNAzyme for developing biosensors for Pb2+. The activity of DNAzymes is often affected by pH and salt, which may in turn affect the sensitivity of related sensors. Although pH can be readily controlled by using a buffer, the effect of salt is more complex. To have a systematic understanding, we herein measured the cleavage activity of GR5 in various concentrations of Na+ and Mg2+. Both metals inhibited the DNAzyme with Pb2+, and the inhibition constants were 1.8 mM Mg2+ and 33.4 mM Na+. For anions, F- inhibited GR5 more strongly than Br-, while Cl- was the least inhibiting anion, which was consistent with the solubility of their lead salts. The reaction can work similarly in many Good's buffers, while phosphate buffer should be avoided. Finally, GR5 is an optimal sequence based on the truncation and elongation studies. This study reveals important solution conditions that should be considered when designing and testing related biosensors for metal detection.