Misfolding of a DNAzyme for ultrahigh sodium selectivity over potassium

Abstract

Herein, the excellent Na+ selectivity of a few RNA-cleaving DNAzymes was exploited, where Na+ can be around 3000-fold more effective than K+ for promoting catalysis. By using a double mutant based on the Ce13d DNAzyme, and by lowering the temperature, increased 2-aminopurine (2AP) fluorescence was observed with addition of both Na+ and K+. The fluorescence increase was similar for these two metals at below 10 mM, after which K+ took a different pathway. Since 2AP probes its local base stacking environment, K+ can be considered to induce misfolding. Binding of both Na+ and K+ was specific, since single base mutations could fully inhibit 2AP fluorescence for both metals. The binding thermodynamics was measured by temperature-dependent experiments revealing enthalpy-driven binding for both metals and less coordination sites compared to G-quadruplex DNA. Cleavage activity assays indicated a moderate cleavage activity with 10 mM K+, while further increase of K+ inhibited the activity, also supporting its misfolding of the DNAzyme. For comparison, a G-quadruplex DNA was also studied using the same system, where Na+ and K+ led to the same final state with only around 8-fold difference in Kd. This study provides interesting insights into strategies for discriminating Na+ and K+.

Global Folding of a Na+‐Specific DNAzyme Studied by FRET

“…The Na + data seem to be able to fit into a simple binding model, whereas the K + data need a two‐stage folding to fit. This is consistent with the misfolding model for K + . Given the very small signal change, we do not further discuss this quantitatively.…”

“…It is likely that other end‐distance‐dependent sensing mechanisms will fail. We observed more differences between the metals if 2AP was used to probe local folding, whereas the largest difference was observed from cleavage activity …”

“…We also titrated Li + and K + into the system (Figure B), and the spectra barely changed either. Based on our previous 2AP probing, Li + cannot fold the DNAzyme, but K + and Na + certainly induces local folding in the aptamer loop . Therefore, such local folding did not affect the global structure of the DNAzyme; thus indicating rigidity of the system.…”

“…To gain a comprehensive understanding, we also studied NaA43T (Figure B) and EtNa DNAzymes (Figure C). NaA43T is a truncated version of the NaA43 DNAzyme, and it has retained Na + binding activity . The FRET‐based folding was studied by using the end‐labeling strategy in Figure D. If up to 200 m m Na + was titrated into the NaA43T DNAzyme, the FRET signal changed very little (Figure B).…”

“…These two techniques suggest a Na + ‐binding aptamer in the DNAzyme. In addition, we studied its local folding through 2‐aminopurine (2AP) fluorescence, and we concluded that misfolding of Ce13d in the presence of K + could explain its excellent selectivity for Na + over K + measured from the catalytic activity …”