A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect

Abstract

In vitro selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln(3+)): Ho(3+), Er(3+) and Tm(3+). The resulting sequences were aligned together and about half of the library contained a new family of DNAzyme. These DNAzymes have a simple loop structure, and they are active only with the seven heavy Ln(3+). Among the tested non-lanthanide ions, only Y(3+) induced cleavage and even Pb(2+) failed to cleave, suggesting a very high specificity. A representative DNAzyme, Tm7, has a sigmoidal metal binding curve with a Hill coefficient of 3, indicating that three metal ions are involved in the catalytic step. Its pH-rate profile has a slope of 1, suggesting a single deprotonation step is involved in the rate-limiting step. Tm7 has a cleavage rate of 1.6 min(-1) at pH 7.8 with 10 μM Er(3+). Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd(2+) alone, or by a mixture of Er(3+) and Cd(2+), suggesting that two interacting metal ions are involved in direct bonding to both non-bridging oxygen atoms. A new model involving three lanthanide ions is proposed based on this study. A biosensor is engineered using Tm7 to detect Dy(3+) down to 14 nM.

Figure 1.

(A) The DNA library sequence. The cleavage site is indicated by the arrowhead. (B) A simplified scheme of the in vitro selection process. Three independent selections with the different Ln³⁺ were carried out. PAGE means gel electrophoresis and two PCR steps were used in step 3 to amplify and re-generate the library. (C) Selection progress and the round 6 library was sequenced. Ln³⁺ concentration was 50 μM for rounds 1–4 and 10 μM for rounds 5–7. Incubation time was maintained at 60 min. (D) Sequence alignment in the enzyme loop region. (E) The secondary structure of the trans-cleaving version of the Tm7 DNAzyme.

Figure 2.

(A) Secondary structures of six DNAzymes in this study. Gel images showing cleavage activity of (B) Tm7 and (C) Ho11 with different Ln³⁺ ions (10 μM) after 1 h reaction. (D) Quantification of the cleavage results of the six DNAzymes by Ln³⁺.

Figure 3.

(A) Fraction of substrate cleavage by the Tm7 DNAzyme using 10 or 100 μM metal ions. Inset: gel image of cleavage in the presence of 10 μM metal ions. The lanes correspond to the metal ions in the x-axis. Only Au³⁺ produced streaking and Er³⁺ and Y³⁺ produced cleavage. (B) Kinetics of Tm7 cleavage at a few Er³⁺ concentrations. (C) Cleavage rate as a function of Er³⁺ concentration. Inset: the same data plotted using the log scale.

Figure 4.

The pH-rate profile of the Tm7 DNAzyme over (A) a wide pH range and (B) the initial linear range. A slope of 0.97 is obtained in (B), indicating a single deprotonation step.

Figure 5.

(A) Structures of the normal phosphate linkage (PO), and the two diastereomers of the PS modification. (B) Cleavage of the Tm7 DNAzyme with the PS-modified substrate (racemic mixture) in the presence of various divalent metal ions and Er³⁺. (C) Cleavage of Tm7 with the PS substrate using a mixture of Er³⁺ and Cd²⁺. (D) Kinetics over 24 h for Tm7 cleaving the PO (black dots), and PS (red triangles) substrate in the presence of 10 μM Er³⁺. The green squares are the Tm7/PS complex incubated without Er³⁺. Inset is the re-plot of the PS sample magnifying the initial kinetics.

Figure 6.

Proposed mechanism of the lanthanide-induced RNA cleavage for the Tm7 DNAzyme. The red arrow indicates nucleophilic attack of the phosphorus center. The bridging oxygen atoms linking the Er³⁺ ions are originated from deprotonated water.

Figure 7.

(A) Sensor response to 0.5 μM of divalent and trivalent metal ions. The list of the other metal ions tested can be found in Figure 2B. (B) Sensor response to 0.5 μM of various Ln³⁺. (C) Sensor signaling kinetics in the presence of various concentrations of Dy³⁺. DNAzyme sensor concentration = 50 nM. (D) Quantification of Dy³⁺ based on the initial rate of sensor fluorescence enhancement. Inset: the initial linear response at low Dy³⁺ concentrations.