Metal-ion detection and speciation analysis is crucial for environmental monitoring. Despite the importance of lanthanides, few sensors are available for their detection. DNAzymes have been previously used to detect divalent metals, while no analytical work was carried out for trivalent and tetravalent ions. Herein, in vitro selection was performed using a Ce(4+) salt as the target metal, and a new DNAzyme (named Ce13) with a bulged hairpin structure was isolated and characterized. Interestingly, Ce13 has almost no activity with Ce(4+) but is highly active with all trivalent lanthanides and Y(3+), serving as a general probe for rare earth metals (omitting Sc). A DNAzyme beacon was engineered detecting down to 1.7 nM Ce(3+) (240 parts per trillion), and other lanthanides showed similar sensitivity. The feasibility of metal speciation analysis was demonstrated by measuring the reduction of Ce(4+) to Ce(3+).

In vitro selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln3+): Ho3+, Er3+ and Tm3+. 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 Ln3+. Among the tested non-lanthanide ions, only Y3+ induced cleavage and even Pb2+ 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 Er3+. Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd2+ alone, or by a mixture of Er3+ and Cd2+, 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 Dy3+ down to 14 nM.

A trivalent lanthanide (Ln 3+ )-dependent RNAcleaving DNAzyme, Ce13d, was recently isolated via in vitro selection. Ce13d is active in the presence of all Ln 3+ ions. Via introduction of a single phosphorothioate (PS) modification at the cleavage site, its activity with Ln 3+ decreases while all thiophilic metals can activate this DNAzyme. This property is unique to Ce13d and is not found in many other tested DNAzymes. This suggests the presence of a well-defined but general metal binding site. Herein, a systematic study of Ce13d with the PO substrate (using Ce 3+ ) and the PS substrate (using Cd 2+ ) is performed. In both the PO and PS systems, the highest activity was with ∼10 μM metal ions. Higher concentrations of Ce 3+ completely inhibit the activity, while Cd 2+ only slows the activity. A comparison of different metal ions suggests that the role of metal is to neutralize the phosphate negative charge. Both systems follow a similar pH−rate profile with a single deprotonation step, indicating similar reaction mechanisms. The activity difference between the R p and S p form of the PS substrate is <10-fold, which is much smaller than most known RNA-cleaving enzymes. Mutation studies identified eight highly conserved purines, among which the two adenines play mainly structural roles, while the guanines are likely to be involved in metal binding. Ce13d can serve as a model system for further understanding of DNAzyme biochemistry and bioinorganic chemistry.

. We previously reported a DNAzyme named Ce13d, which has similar responses to all the trivalent lanthanides. Combining these two allows for a ratiometric assay that identifies a few large lanthanides.

Chromium is a very important analyte for environmental monitoring, and developing biosensors for chromium is a long-standing analytical challenge. In this work, in vitro selection of RNAcleaving DNAzymes was carried out in the presence of Cr 3+ . The most active DNAzyme turned out to be the previously reported lanthanide-dependent Ce13d DNAzyme. While the Ce13d activity was ~150-fold lower with Cr 3+ compared to that with lanthanides, the activity of lanthanides and other competing metals was masked by using a phosphate buffer, leaving Cr 3+ the only metal that can activate Ce13d. With 100 µM Cr 3+ , the cleavage rate is 1.6 h -1 at pH 6.Using a molecular beacon design, Cr 3+ was measured with a detection limit of 70 nM, significantly lower than the U.S. Environmental Protection Agency (EPA) limit (11 μM). Cr(VI) was measured after its reduction by NaBH4 to Cr 3+ , and it can be sensed with a similar detection limit of 140 nM Cr(VI), lower than the EPA limit of 300 nM. This sensor was tested for chromium speciation analysis in a real sample, supporting its application for environmental monitoring. At the same time, it has enhanced our understanding on the interaction between chromium and DNA.3

Trivalent lanthanide ions (Ln3+) were recently employed to select RNA-cleaving DNAzymes, and three new DNAzymes have been reported so far. In this work, dysprosium (Dy3+) was used with a library containing 50 random nucleotides. After six rounds of in vitro selection, a new DNAzyme named Dy10a was obtained and characterized. Dy10a has a bulged hairpin structure cleaving a RNA/DNA chimeric substrate. Dy10a is highly active in the presence of the five Ln3+ ions in the middle of the lanthanide series (Sm3+, Eu3+, Gd3+, Tb3+, and Dy3+), while its activity descends on the two sides. The cleavage rate reaches 0.6 min–1 at pH 6 with just 200 nM Sm3+, which is the fastest among all known Ln3+-dependent enzymes. Dy10a binds two Ln3+ ions cooperatively. When a phosphorothioate (PS) modification is introduced at the cleavage junction, the activity decreases by >2500-fold for both the R p and S p diastereomers, and thiophilic Cd2+ cannot rescue the activity. The pH–rate profile has a slope of 0.37 between pH 4.2 and 5.2, and the slope was even lower at higher pH. On the basis of these data, a model of metal binding is proposed. Finally, a catalytic beacon sensor that can detect Ho3+ down to 1.7 nM is constructed.

Developing chemical probes to distinguish each lanthanide ion is a long-standing challenge. Aside from its analytical applications, solving this problem will also enhance our knowledge in metal ligand design. Using in vitro selection, we previously reported four RNA-cleaving DNAzymes, each with a different activity trend cross the lanthanide series. We herein performed another eight in vitro selection experiments using each and every lanthanide from La 3+ to Tb 3+ but excluding the radioactive Pm 3+ . A new DNAzyme named Gd2b was identified and characterized. By labeling this DNAzyme with a fluorophore/quencher pair to create a catalytic beacon, a detection limit of 14 nM Gd 3+ was achieved. With the same beacon design, all the five lanthanide-specific DNAzymes were used together to form a sensor array. Each lanthanide ion produces a unique response pattern with these five sensors, allowing a pattern-recognition-based linear discriminant analysis (LDA) algorithm to be applied, where separation was achieved between lanthanides and nonlanthanides, light and heavy lanthanides, and for the most part, each lanthanide. These lanthanidespecific DNA molecules are useful for understanding lanthanide coordination chemistry, designing hybrid materials, and developing related analytical probes.

Abstract.Thallium (Tl) is a highly toxic heavy metal situated between mercury and lead in the periodic . This system is engineered into a catalytic beacon for Tl 3+ with a detection limit of 1.5 nM, which is below its maximal contamination limit defined by the US Environmental Protection Agency (10 nM).3