Metal Sensing by DNA - ACS Publications - American Chemical Society

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Metal Sensing by DNA Wenhu Zhou,†,‡ Runjhun Saran,‡ and Juewen Liu*,‡ †

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China Department of Chemistry, Water Institute, and Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

‡

ABSTRACT: Metal ions are essential to many chemical, biological, and environmental processes. In the past two decades, many DNA-based metal sensors have emerged. While the main biological role of DNA is to store genetic information, its chemical structure is ideal for metal binding via both the phosphate backbone and nucleobases. DNA is highly stable, cost-effective, easy to modify, and amenable to combinatorial selection. Two main classes of functional DNA were developed for metal sensing: aptamers and DNAzymes. While a few metal binding aptamers are known, it is generally quite difficult to isolate such aptamers. On the other hand, DNAzymes are powerful tools for metal sensing since they are selected based on catalytic activity, thus bypassing the need for metal immobilization. In the last five years, a new surge of development has been made on isolating new metalsensing DNA sequences. To date, many important metals can be selectively detected by DNA often down to the low parts-per-billion level. Herein, each metal ion and the known DNA sequences for its sensing are reviewed. We focus on the fundamental aspect of metal binding, emphasizing the distinct chemical property of each metal. Instead of reviewing each published sensor, a high-level summary of signaling methods is made as a separate section. In principle, each signaling strategy can be applied to many DNA sequences for designing sensors. Finally, a few specific applications are highlighted, and future research opportunities are discussed.

CONTENTS 1. Introduction 2. General Aspects of Metal Binding by DNA 2.1. Advantages of DNA for Metal Sensing 2.2. Metal Binding Sites in DNA 2.3. Properties of Metal Ions for DNA Recognition 2.4. Aptamers 2.5. DNAzymes 3. Metal-Specific DNA Sequences 3.1. Alkali Metal Ions 3.1.1. Sodium 3.1.2. Potassium 3.1.3. Cesium 3.2. Alkaline Earth Metal Ions 3.2.1. Magnesium 3.2.2. Calcium 3.2.3. Strontium and Barium 3.3. Lanthanide and Actinide Ions 3.3.1. Lanthanide Ions 3.3.2. Uranium (UO22+) 3.4. Post-Transition Metal Ions 3.4.1. Lead 3.4.2. Thallium 3.4.3. Aluminum 3.5. Transition Metals 3.5.1. Copper 3.5.2. Zinc 3.5.3. Mercury 3.5.4. Cadmium © 2017 American Chemical Society

3.5.5. Cobalt 3.5.6. Manganese 3.5.7. Nickel 3.5.8. Iron 3.5.9. Molybdenum and Tungsten 3.5.10. Chromium 3.6. Noble Metals 3.6.1. Silver 3.6.2. Gold 3.6.3. Platinum 3.6.4. Palladium 4. Signaling Methods 4.1. Catalytic Beacons 4.2. Aptamer Beacons 4.3. Aptazymes 4.4. Signaling Based on Local Folding 4.5. Label-Free Methods 4.6. Lateral Flow Devices 5. Emerging Applications 5.1. Metal Speciation 5.2. Environmental Monitoring 5.3. Caged Aptamers/DNAzymes 5.4. Intracellular Sensing and Imaging 5.5. Sensing in Other Biologic Fluids 5.6. Pharmaceutical and Theranostic Applications

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Received: January 24, 2017 Published: June 9, 2017 8272

DOI: 10.1021/acs.chemrev.7b00063 Chem. Rev. 2017, 117, 8272−8325

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Figure 1. Color-coded periodic table showing the current status of research on nucleic acid based metal sensing. This review is focused on the metals coded in green, for which extensive studies have been carried out. The blue ones have been researched on their oxides/complexes for DNA adsorption/binding. The pink ones have been tested by sensor arrays but without specific binding DNA sequences reported or detailed studies. The yellow ones form oxyanions in water (or inert gases) and lack a preferred mechanism of binding to DNA. The gray ones have yet to be studied, and the radioactive elements in purple are not discussed.

5.7. DNA-Functionalized Hydrogels and Liposomes 5.8. Signal Amplification 5.9. DNA Nanotechnology 6. Future Directions 6.1. Improving Sensitivity and Binding Affinity 6.2. Improving Selectivity 6.3. Developing Specific Applications 6.4. Fundamental Studies Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Abbreviations References

To complement instrumentation analysis, efforts have been made to develop metal sensors. Antibodies represent a popular sensing platform. However, metal ions are too small for direct antibody recognition and they have to be chelated first,5 which may compromise selectivity. In addition, antibodies work optimally under physiological conditions, while environmental samples may need to be detected under other conditions. Protein and peptide based metal sensors have also been demonstrated,6,7 but they are prone to irreversible denaturation, a problem common to all proteins. Chemical metal sensors mainly rely on rationally designed fluorescent chelators.8−14 They distinguish metal ions based on their size, charge, and sometimes thiophilicity. A few such probes have been commercialized for important metals such as Ca2+ and Zn2+. Using nucleotides and nucleic acids as metal ligands has been researched for decades, mainly in the context of bioinorganic chemistry15−18 and medicinal chemistry.19,20 Efforts on directed evolution of metal-binding nucleic acids started in the 1990s.21,22 The role of metal ions in ribozyme catalysis has been a key topic from the 1980s to date.23−26 Nucleic acids did not catch much attention for metal detection until 2000,27 but since then, we have witnessed a rapid development.28−32 Compared to DNA, RNA is less stable and has not been researched much for metal sensing, although metal-dependent riboswitches (a fragment of mRNA) in bacterial cells have been identified.33−35 The main function of DNA in biology is for storing genetic information. DNA interacts only with a few metal ions in cells, namely K+, Na+, and Mg2+, while the concentrations of other free metals are too low to be important (far below their Kd’s for DNA). Natural DNA is a duplex with its nucleobases shielded by the phosphate backbone. Therefore, it is difficult to link such DNA to specific metal recognition. With single-stranded oligonucleotides, the bases can also contribute to metal coordination by forming 3D binding pockets. Over the past two decades, many DNA sequences have been reported with

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1. INTRODUCTION Metal ions play key structural and functional roles in biology. Understanding the distribution and concentration fluctuation of metal ions in biology is a central topic in bioanalytical chemistry with applications in cell signaling, medicine development, and enzyme catalysis.1,2 At the same time, anthropological activities have mobilized huge quantities of metal species through mining, discharging industrial water, corrosion, coal burning, and waste dumping, leading to serious environmental and health problems.3 Therefore, metal detection is an important analytic task for biological and environmental applications. So far, the standard methods for metal analysis rely on instruments such as atomic absorption/emission and mass spectroscopy.4 These techniques are highly accurate and sensitive with industrial standards, but they are costly, available only in large centralized laboratories and require extensive sample pretreatment, disallowing for on-site, real-time, or in situ measurement. 8273


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Figure 2. Schemes of (A) diffuse electrostatic, (B) outer-sphere, and (C) inner-sphere metal binding to a phosphate. Redrawn from ref 71. (D) Nucleotide coordination affinity to various divalent metal ions. Figure adapted with permission from ref 17. Copyright 2010 American Chemical Society. The numbers listed are the log of Ka. Schemes of (E) metal binding by G-quadruplex DNA, (F) Ag+ by cytosine, and (G) Hg2+ by thymine. (H) Structures of a normal phosphodiester (PO) and a phosphorothioate (PS) linkage.

remarkable metal binding affinity and specificity.31 An impressive fraction of metals in the periodic table can now be sensed by DNA (green and pink spots in Figure 1). A biosensor contains a biomolecule for target recognition (in this case DNA) and a signal transduction element. DNA-based biosensors have been reviewed previously in general36−46 and also specifically for metal ions.31,47 The latest comprehensive reviews are already more than 5 years old. Most previous reviews focused on the sensor design aspect, using only a few model sequences (e.g., DNA specific for Pb2+, Hg2+, Ag+, and K+ being the most popular) to improve signal transduction. Over the past 5 years, much progress has been made in discovering new DNA sequences for metal sensing. Given recent developments, we herein focus on the metal recognition aspect (e.g., new DNA sequences for metal binding) with extensive mechanistic discussion, which may contribute to new analytical applications. To be comprehensive, this review covers DNA sequences reported since the 1990s. In addition to DNA, relevant metalspecific RNAs are also discussed. We only review DNA binding to free metal ions, while metal complexes are not covered. Complexes containing Ru(II), Os(II), Rh(III), and Ir(III) have excellent DNA binding and luminescence properties, and interested readers are referred to specific reviews on this topic.48−53 This review is comprised of the following four parts. First, the basic interactions between metal ions and nucleotides/ DNA are introduced from a bioinorganic chemistry standpoint. Some classical methods for obtaining metal-binding DNA sequences are also discussed. Second, each metal ion is reviewed individually for their specific DNA interactions. The third part describes representative signaling methods. Since the same sensor design can be used by many DNA sequences, we often outline signaling methods without specifying particular DNA sequences. Finally, a few emerging applications are discussed together with future research opportunities.

2. GENERAL ASPECTS OF METAL BINDING BY DNA 2.1. Advantages of DNA for Metal Sensing

DNA has a number of desirable properties for metal sensing. First, DNA is a polyanion allowing electrostatic attraction with metal ions. DNA phosphates can bind hard/borderline metals, while various bases coordinate with metal ions with different affinities.15,17,54 Through tertiary DNA folding, 3D binding pockets can form to accommodate specific metal coordination preferences, which is difficult to achieve via rational ligand design. Such versatile binding modes give DNA the potential for selective metal binding. Second, DNA is highly stable and can be renatured after denaturation without losing its metal binding affinity. Third, chemical synthesis of DNA is currently at a low cost with a diverse range of modifications available at essentially any site of choice. DNA is a highly programmable molecule, which is exemplified by the remarkable advancement in its nanotechnology applications.55 Fourth, sensors can be designed largely based on the secondary structure of DNA with minimal knowledge of its tertiary folding. For comparison, it is much more difficult to predict the structure of peptides and proteins based on their primary sequences. Finally, and most importantly, DNA is amenable to in vitro selection, allowing combinatorial searches for metal binding sequences. Such selections can be carried out in most laboratories with common equipment. 2.2. Metal Binding Sites in DNA

Metal coordination by DNA has been extensively studied.15,17,54 At the simplest level, metal ions are treated as point charges diffusing around DNA polyanions by pure electrostatic interactions (Figure 2A). Metal binding to the phosphate backbone stabilizes the DNA duplex (e.g., increasing DNA melting temperature, Tm). However, this pure electrostatic picture disregards the chemical nature of metal ions and DNA. For example, group 1A and 2A metals mainly interact with DNA phosphate, and duplex DNA stability is retained even with 1 M Na+ or Mg2+.56 However, the first row transition metals, Cd2+, Pb2+, and trivalent lanthanides, interact with both phosphate and bases. They start to destabilize duplex DNA 8274


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beyond a few μM since they can coordinate with DNA bases, thus disrupting internucleobase hydrogen bonds.57 Even softer metals such as Ag+ and Hg2+ mainly interact with DNA bases.54 Some metals such as platinum and chromium bind to DNA bases very strongly and essentially irreversibly under ambient conditions.58 The structures of deoxyadenosine monophosphate and the other three nucleobases of DNA are shown in Figure 2D. We summarize a few properties of nucleotides for metal coordination. (1) All of the nucleobases are charge neutral at physiological pH. The N1 position of adenine can be protonated, and its protonated form has a pKa of 3.5. The N3 position of cytosine can also be protonated with a pKa of 4.2. After protonation, it becomes more difficult for these sites to bind metal. The N3 position of thymine needs to deprotonate with a pKa of 9.9 before it can coordinate to a metal. However, with a very strong interaction, Hg2+ can displace this proton at neutral or even slightly acidic pH.59 The N7 position of guanine can be protonated with a pKa of 2.1. Therefore, this site is available for metal coordination in most conditions. In general, the binding affinity with transition metals follows the order of N7 (guanine) > N3 (cytosine) > N7 (adenine) > N1 (adenine) > N3 (adenine, guanine).15 (2) The exocyclic amine groups in nucleobases are poor metal ligands since the lone pair electron delocalizes into the aromatic rings. (3) Each phosphate carries one negative charge with a pKa below 2. Therefore, DNA is highly negatively charged at physiological pH. Phosphate prefers metals with a high charge and small size (e.g., hard metals with a high charge density). The electrostatic repulsion between phosphate groups is a main force to destabilize DNA structures, and such repulsion can be alleviated by metal ions. (4) The deoxyribose ring contributes little to metal coordination. In certain cases, the ribose in RNA can bind metal complexes (e.g., Os(VI)).60,61 The use of nucleotides as metal ligands has been extensively studied.15,17,62−64 For example, Sigel and Sigel summarized the metal binding affinity of some typical nucleotide sites (Figure 2D),17 such as the phosphate, the N7 of guanine and adenine, and the N3 of cytosine. Overall the affinities are quite weak for the individual sites (e.g., association constant, Ka < 500).17 Stability is increased by the chelation effect. For example, AMP binds Cu2+ with a Ka of ∼1500 by using both the phosphate and adenine.17 With a few nucleotides involved, the affinity is further enhanced (e.g., the pGpG dinucleotide binds Pb2+ with a Ka of ∼104).17 In addition, a few specific binding mechanisms have been discovered, such as Hg2+/thymine,65,66 Ag+/ cytosine,67 and Pb2+/guanine quadruplex68 interactions (Figure 2E−G). Metal binding affinity can also be altered by introducing chemical modifications, such as by replacing one of the nonbridging oxygen atoms in the phosphate with sulfur producing a phosphorothioate (PS) linkage (Figure 2H). With a PS, the affinity of the phosphate to soft metals is increased while its binding to hard metals is weakened.69 Numerous modifications to the bases are also possible, such as adding imidazole, ammonium, and guanidinium groups.70

with a tightly coordinated water shell. As such, the size of hydrated metal ions is more relevant. In many cases, however, strong ligands can displace one or more of the coordinated water molecules. This process is associated with entropy gain due to the released water but usually absorbs heat for water dissociation. Direct metal binding is called inner-sphere coordination (Figure 2C), while water mediated binding is called outer-sphere coordination (Figure 2B). For metals in the same group of the periodic table, heavier ions have a lower charge density and thus weaker affinity for water binding. This leads to a smaller hydrated size than the lighter metals. For example, the ionic radius of Li+ (0.68 Å) is smaller than that of Cs+ (1.69 Å), but the hydrated ion radius is larger for Li+ (6 Å) than that for Cs+ (2.5 Å) (Table 1). A classic example of ligand Table 1. Size and Solvation Enthalpy of Alkali and Alkaline Earth Metal Ions74,75 ion

Pauling diametera DP (Ǻ )

hydrated diameterb DH (Ǻ )

solvation enthalpyc −ΔHS (kJ mol−1)

Li+ Na+ K+ Rb+ Cs+ Be2+ Mg2+ Ca2+ Ba2+

1.36 1.90 2.66 2.96 3.38 0.62 1.30 1.98 2.70

12.0 8.0 6.0 5.0 5.0 16.0 16.0 12.0 10.0

530 420 340 315 280 2520 1960 1615 1340

a

Pauling diameter (DP). Dp = 2Rp, where RP is the crystal radius of the bare ion. bHydrated diameter (DH). DH = 2RH, where RH is the effective radius of a hydrated ion in solution. cSolvation enthalpy (ΔHS) measures the interaction between an ion and solvent molecules. A small −ΔHS value indicates a weaker hydration shell and the hydration ion is easily dehydrated.

design based on metal size is the crown ethers.72 In nucleic acids, a G-quadruplex is selectively stabilized by K+ but not by Li+, which is partially attributable to size matching.73 (2) pKa of metal bound water. Water has a pKa of 14 at 25 °C. When a water molecule is associated with a metal ion, it often becomes more acidic especially when the metal ion is in a high oxidation state. As such, metal bound water molecules can release a proton and become a metal bound hydroxyl group, a process known as hydrolysis. For example, Mg2+ has a pKa value of 11.42, lower than that of Ca2+ (12.7), but much higher than those of Zn2+ (8.96) or Pb2+ (7.8).76 This indicates that Mg2+ interacts with the water more strongly than Ca2+ due to its higher charge density but weaker than Pb2+ and Zn2+. After deprotonation, the metal bound hydroxyl can act as a general base to accept protons, which is important for the RNA cleavage reaction (vide infra). Another consequence of hydrolysis is decreased metal charge. Metal ions with a high oxidation state are particularly prone to hydrolysis. Sometimes extensively hydrolyzed metals (e.g., much beyond the first pKa) even become anions such as MnO4− and are then repelled by DNA. In other cases, hydrolysis leads to further condensation of metal to produce metal hydroxides or oxides, which is particularly prevalent in group 3A and 4A metals. Unless pH is sufficiently low to suppress hydrolysis, their interaction with DNA can hardly be probed as free metal ions. The pKa of metal bound water has been used to explain the activity trend of some RNA-cleaving DNAzymes.77−79

2.3. Properties of Metal Ions for DNA Recognition

Designing selective metal ligands is a long-standing challenge of inorganic chemistry. In general, metal ions have the following properties that can be harnessed for their detection. (1) Size. Size is a fundamental property of metal ions useful for ligand design. In aqueous solutions metal ions are hydrated 8275


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Ag+ (Figure 2F)67 and thymine for Hg2+ (Figure 2G), allowing rational aptamer design for these metals.59,65 However, for most other metals, their aptamers have to be obtained by combinatorial selection. The process of isolating aptamers is called systematic evolution of ligands by exponential enrichment (SELEX).88,89 SELEX starts with a huge DNA library containing 1013 to 1016 random sequences. Those that can bind to the target are collected and amplified by the polymerase chain reaction (PCR). Typically, aptamer selection requires immobilization of target molecules, but it is nearly impossible to immobilize metal ions without significantly masking their coordination sites. While a few selections were carried out with immobilized metals such as Zn2+ and Ni2+,21,90 the resulting aptamers were not followed up for analytical applications, and they do not bind metals strongly or selectively. To overcome the metal immobilization problem, an alternative method was developed by immobilizing the DNA library via a short duplex region (typically 12 base pairs, Figure 3). If a sequence can bind the target metal (in this case Zn2+)

(3) Lewis acidity. Metal ions are electron deficient, and they can generally accept electron pairs from ligands and thus act as Lewis acids. In this context, the hard soft acid base (HSAB) theory is a good starting point for predicting and controlling metal/ligand interactions. The phosphate linkages in nucleic acids are a hard Lewis base to interact favorably with hard metals such as Mg2+. When soft metals such as Cd2+ and Hg2+ are used, their phosphate binding is weak. Binding to these soft metals can be promoted by introducing softer PS substitutions, which is commonly used to probe metal binding in ribozymes. If a PS modification shifts the metal preference from hard to soft in a DNAzyme/ribozyme, it usually suggests inner-sphere coordination at that particular sulfur/oxygen.69,71,80,81 (4) Charge. Charge is another fundamental property for distinguishing different metals. Since DNA is a polyanion, the number of charges on metal ions is critical for DNA binding. In general, the effects of group 1A metals are studied at above 10 mM, and group 2A metals at ∼1 mM, while trivalent lanthanide ions at ∼10 μM, highlighting the importance of charge. For metals in other groups, the interaction may be complicated by metal hydrolysis and DNA base binding, where metals with a high oxidation state may not necessarily have a stronger electrostatic interaction. (5) Coordination preference. For transition metal ions with d orbitals, extra stabilization energy from the ligand field and splitting of the d orbitals can contribute to metal coordination. As such, transition metals often bind to DNA more strongly than group 1A and 2A metals. A good example is that Hg2+ prefers linear coordination and the B-form DNA with a thymine−thymine mismatch can satisfy it, explaining its affinity and selectivity for Hg2+ over other metal ions.82 It is quite possible that single-stranded DNA can fold into 3D structures to achieve optimal metal coordination. In most cases, however, such coordination geometry of DNA remains elusive due to a lack of structural biology data. Only very limited structures have been solved by X-ray crystallography or NMR, mainly Gquadruplex DNA,83 Hg2+-thymine containing duplex DNA,84 and, very recently, a DNAzyme.85 (6) Ligand exchange rate. Most common metal ions have relatively fast ligand exchange rates in water (e.g., >1 s−1).86 Therefore, on the time scale of human operation (e.g., >10 s), we assume that the binding equilibrium is reached. This allows us only to consider binding thermodynamics without paying attention to the kinetic aspect. However, a few important metals have very slow ligand exchange rates. For example, Pt2+ binds to DNA nearly irreversibly due to a very high activation energy of ligand dissociation (ligand exchange rate Ni2+ > Co2+ > Mg2+.57 This simple melting trend is quite informative. For example, Cu2+ has a very strong destabilizing effect, suggesting its strong interaction with DNA bases. 3.1. Alkali Metal Ions

The alkali metals refer to Li+, Na+, K+, Rb+, and Cs+. Table 1 lists their ionic radii and hydration energies, which can directly affect their interaction with DNA. These cations have long been used as general buffer salts to screen charge repulsion in DNA. A few techniques have been used to quantitatively study their binding to DNA, including molecular dynamics simulation,120 nuclear magnetic resonance (NMR),121,122 atomic force microscopy (AFM),123 electrochemistry,124 and crystallography.125 However, the general trend of interaction among them has been quite inconsistent and even controversial in different reports, which is likely due to different experimental systems and characterization techniques.126 DNA Tm, however, is often higher in the presence of Li+ than with other metals in this 8278


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Figure 6. (A) Typical structures of intramolecular (A) parallel and (B) antiparallel G4. Location of (C) Na+ and (D) K+ in G4 structures, where Na+ and K+ are indicated by the purple and green spheres, respectively.151

demonstrated due to a high background ionic strength inducing nonspecific DNA condensation and a high intracellular K+ concentration for competition. 3.1.2. Potassium. Using DNA for K+ detection relies mainly on G4 DNA. G4 is a noncanonical nucleic acid structure with stacked guanine tetrads assembled by Hoogsteen hydrogen bonding, in which K + coordinates the O6 of guanines.144,145 Two or more such G-tetrads connect and stack together to form a G4 structure. Many G4-forming sequences were found in genome DNA and mRNA in cells, which may regulate replication, transcription, and translation.146−149 G4 structures can be formed by unimolecular, dimolecular, and even tetramolecular sequences with parallel (Figure 6A) or antiparallel orientation (Figure 6B). The relative strand orientation can be analyzed by circular dichroism (CD) spectroscopy.150 X-ray crystallography shows that metals are located quite differently in the central channel of G4 depending on the size of metal ions.144,151,152 For example, Na+ is smaller and sits in the plane of a G-tetrad (Figure 6C), whereas the larger K+ lies between two G-tetrads (Figure 6D). Therefore, each K+ is coordinated by eight O6 groups from two consecutive stacked G-tetrads. The metal preference of G4 DNA follows the order of K+ > Na+ > Li+,153,154 and favored binding of K+ over Na+ is attributable to the lower dehydration energy of K+.155 This also suggests that the binding is through direct metal coordination without water intervening. On the other hand, the size of Li+ is too small, which may explain its weak binding to G4 DNA.73 The ionic size also determines the movement of these cations through G4 DNA channels.156 For example, the larger K+ must squeeze through a channel to reach the adjacent binding site, while Na+ diffuses continuously throughout the channel. The kinetics of K+- and Na+-induced G4 folding were studied by Chaires and co-workers using three model human telomeric oligonucleotides.157 The results suggest that the folding is a multistep process with a rapid formation of intermediate cationoligonucleotide complexes followed by slower isomerizing

state phosphorane. In the NaA43, a nucleobase might substitute the role of Ce3+. Recently, we reported another Na+-specific DNAzyme named EtNa with a completely different structure (Figure 5C).136 Its rate is slightly slower (0.06 min−1) with much weaker Na+ binding affinity in water (e.g., Kd > 1 M Na+), but it is highly selective for Na+ (Figure 5D, lower panel). With ∼50% ethanol, the EtNa has a Kd of 22 mM Na+. Therefore, it is more suitable for applications involving organic solvents. It is quite remarkable that DNA can recognize Na+ with such high specificity. Compared with many small molecule probes with Kd > 100 mM Na+,12,13 these DNAzymes bind Na+ with a reasonably strong affinity. Using sensitized Tb3+ luminescence, DMS footprinting, and 2-aminopuine, a Na+ binding aptamer was identified from the Ce13d DNAzyme with a Kd of ∼20 mM Na+.134,137,141 It is likely that a similar aptamer also exists in the NaA43. This aptamer, however, requires the full DNAzyme structure of the Ce13d for Na+ binding, and all the attempts to truncate the sequence led to abolished Na+ binding. Interestingly, no such aptamer was found in the EtNa, and it binds Na+ through a distinct mechanism. The NaA43 is superior for intracellular Na+ detection since it works with Na+ alone, while the Ce13d provides a useful aptamer scaffold for designing reversible sensors for dynamic monitoring. The EtNa is only useful in organic solvents since it is too slow in water albeit with excellent selectivity for Na+. Na+ can also stabilize G4 structures, but it is usually less efficient than K+ for this purpose.142 Nevertheless, G4 structures with Na+ specificity were reported for Na+ sensing by Tang et al.143 They used a G4 sequence called p25, forming a hybrid-type conformation with K+ but an antiparallel conformation with Na+. These two conformations have different binding affinities toward hemin, and thus the catalytic activity of the resulting hemin-p25 DNAzymes with peroxidaselike activity was used for Na+ sensing. However, such sensors for detection Na+ in biological samples have yet to be 8279


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Figure 7. (A) Structures of guanine and isoguanine. (B) A tetraplex stabilized by monovalent cations,186,187 and (C) a pentaplex stabilized by Cs+ based on isoguanines.188

Table 2. Parameters Summarizing the Binding Alkaline Earth Metals with DNA Phosphate and Nucleobases ion

ΔTm (°C)a

isolated guanineb ΔE (kcal/mol)d

isolated adenineb ΔE (kcal/mol)d

guanine in duplexb ΔE (kcal/mol)d

guanine in duplexc ΔE (kcal/mol)d

adenine in duplexb ΔE (kcal/mol)d

adenine in duplexc ΔE (kcal/mol)d

Mg2+ Ca2+ Sr2+ Ba2+

+8.1 +3.7 +7.8 +5.7

−206.0 −136.6 −116.6 −120.3

−112.2 −66.7 −53.2 −55.5

−198.7 −133.9 −116.6 −118.8

−89.8 −86.5 −80.1 −70.8

−107.9 −61.5 −48.9 −51.4

−46.0 −33.5 −28.9 −28.1

ΔTm is a measure of Tm increase of a 160 bp calf thymus DNA (55 mg/mL) after addition of 100 mM divalent metal chloride.198 bThe interaction of bare metals with DNA bases. cThe interaction of hydrated metals with DNA bases. dΔE is the interaction energy evaluated at the Mùller-Plesset second-order (MP2) perturbation theoretical level using the function counterpoise method.194

a

steps. For K+-induced folding, a single isomerization was observed with a relaxation time τ of 20−60 ms depending on the sequence. Na+-driven folding, in contrast, consists of three exponentials with the τ-values of 40−85 ms, 250−950 ms, and 1.5−10.5 s, respectively, indicating three isomerization steps. It appears that the folding pathway in the presence of K+ is simpler than that in Na+. The binding of K+ toward G4 DNA is highly cooperative, and the binding constant is quite different among various DNA sequences. For example, the Kd of K+ to some human telomere sequences is 0.5−1 mM,157 while only 5 μM for the thrombin aptamer.158 Aside from K+ and Na+,143 many other cations are also able to stabilize G4 structures with distinct folding topologies, such as Rb+,159 NH4+,160 Tl+,161−163 and divalent ions (e.g., Sr2+, Ba2+, and Pb2+).164−169 This hints an intrinsic disadvantage of using G4 DNA for K+ detection. Nevertheless, many G4 DNAs have been converted into sensors for K+ detection in water samples with a relatively simple metal composition.170−179 To realize its biological applications, several efforts have been made to increase selectivity. Some studies showed nanomolar K+ sensitivity with ∼104-fold selectivity over Na+.180,181 With these progresses, using G4 DNA for K+ detection in serum and urine has been achieved.180−183 However, since G4 can respond to many other metals, novel DNA sequences that can better discriminate K+ from Na+ and other ions are still to be developed. 3.1.3. Cesium. Cs+ is mildly toxic because of its similarity to + K . Cs+ can substitute K+ in some biological processes, causing K+ starvation.184 The first report of Cs+-specific DNA was only seen recently. Ma and co-workers found that a G-pentaplex formed by an isoguanine-rich DNA selectively binds Cs+.185 Isoguanine is a natural guanine analog in which the carbonyl and amino groups are swapped (Figure 7A). Akin to guanine,

isoguanine is able to form G4 in the presence of monovalent cations (Figure 7B).186,187 Interestingly, this guanine isomer can also fold into a pentaplex stabilized only by Cs+ (Figure 7C), which was utilized for Cs+ detection.188 3.2. Alkaline Earth Metal Ions

Alkaline earth metals (group 2A) contain Be2+, Mg2+, Ca2+, Sr2+, Ba2+, and Ra2+. Among them, Mg2+ and Ca2+ are the most important in biology, while Be2+ is toxic,189 Ba2+ is used to help X-ray imaging, and Ra2+ is radioactive. The size and dehydration energy of these metals follow the same trend as those of alkali metals (Table 1). The phosphate backbone of DNA is the main binding site for these metals,190,191 and DNA base interactions were also reported in several cases. For example, the X-ray crystallography studies of Z-DNA indicated that Ba2+ coordinates to the O6 and N7 atoms of two guanines to bridge two side-by-side Z-DNA helices.192 Mg2+ binds to the O6 and N7 of guanine through its hydration shell (e.g., outersphere interactions).193 The interactions of these metals with nucleobases were also studied by using both isolated bases and DNA duplexes (Table 2).194−197 First, these metals prefer binding to guanine and adenine bases, and the binding energies with guanine are systematically larger than those with adenine. This is likely due to the larger dipole moment of guanine, as well as the two coordination sites for guanine (O6 and N7) versus one for adenine (N7). Second, the binding energies gradually reduce along the group 2A metals, and the bare metals have much higher interaction energies than hydrated metals. Finally, the amount of Tm increase gradually decreases with larger group 2A metals except for Ca2+.198 For specific binding, functional DNAs that can recognize Mg2+, Ca2+, Sr2+, and Ba2+ have been reported. 8280


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Figure 8. (A) Six water molecules coordinating to Mg2+ forming [Mg(H2O)6]2+ with an octahedral geometry. (B) Chemical structures of nucleotides and the normalized interaction frequency of each RNA atom for inner-sphere or outer-sphere Mg2+ binding. The atoms with high Fatom values are highlighted in green.209 An example of Mg2+ binding to (C) phosphate oxygen with inner-sphere coordination and (D) to guanine O6/N7 through outer-sphere hydrogen bonds.206

3.2.1. Magnesium. Mg2+ is the major intracellular divalent cation important for almost all nucleic acid related biological processes.63,199,200 Mg2+ is presumed to be an essential cofactor for most ribozymes.25,201 In aqueous solutions, Mg2+ binds six water molecules in an octahedral arrangement to form [Mg(H2O)6]2+ (Figure 8A). The first pKa of these waters is 11.4,202 rendering them as good hydrogen-bond donors. Mg2+ has a much higher dehydration enthalpy compared to other alkaline earth metals (Table 1). As a result, its ligand exchange rate is relatively slow, often making it bind via outer-sphere coordination.203 In several cases, strong ligands (e.g., G-O6/ U−O4 carbonyl groups and backbone phosphate) may displace 1−3 water ligands in [Mg(H2O)6]2+, forming inner-sphere coordination.204−206 High-resolution X-ray structures of the Bform DNA revealed that hydrated Mg2+ ions mainly reside near the phosphate groups.207 A subsequent molecular dynamics simulation suggests that Mg2+ selectively locates at the phosphate backbone as well as the major groove of G/C bases.208 Zheng et al. mapped the Mg2+-binding architectures in RNA using the RNA crystals in the Protein Data Bank (PDB) (Figure 8B).209 They evaluated the relative frequency of each atom in RNA that binds Mg2+ (Fatom) normalized by the frequency of all type of atoms.207 The most frequent binding ligand is the nonbridging oxygens at the phosphate (total Fatom = 13.9), attributable to their strong electrostatic attraction. For

such binding, 66% of Mg 2+ is through inner-sphere coordination (Figure 8C).206 For nucleobases, the major binding sites are A-N7, U-O4, G-N7, and G-O6, which in general prefer to outer-sphere binding (Figure 8D) with innersphere interactions also observed except for A-N7. Compared to nitrogen ligands, oxygen ligands are more likely to bind via inner-sphere coordination. Overall, the frequency for nucleobases binding Mg2+ in the inner-sphere manner follows G > U > A > C, while for outer-sphere binding, the trend is G > A > U > C. Leonarski et al. re-examined the inner-sphere binding of Mg2+ to imine N1/N3/N7 atoms in RNA, DNA, and purine containing metabolites, also concluding that the occurrences of inner-sphere binding to these atoms are much less frequent than previously presented.210 They attributed this to the assignment of electron density of Na+, K+, NH4+, polyamines, or water to Mg2+. They also claimed that Mg2+ rarely binds to purine N7 sites through inner-sphere coordination. Mg2+ is the most studied metal in ribozyme catalysis, especially for the hammerhead ribozymes (HHRzs) due to its small size.211−213 The extensively truncated minimal HHRzs require a very high Mg2+ concentration for activity (much more than intracellular Mg2+ of ∼1 mM). To be active at the physiological Mg2+ concentration, the peripheral loops need to form tertiary stabilizing motifs.214−216 No one has explored 8281


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other active sequences.231,232 Overall, none of the isolated DNAzymes are highly specific for Mg2+, rendering them unsuitable for sensing Mg2+. To be successful, novel selection strategies are needed to inhibit the 8-17 motif.233,234 3.2.2. Calcium. Ca2+ is an essential nutrient accounting for ∼2% of the total body weight. Aside from its structural role in skeletal and soft tissues, Ca2+ is directly involved in a wide range of biological processes and its concentrations are strictly regulated.235 For example, Ca2+ in serum is about 2.5 mM, while intracellular Ca2+ exists only at a trace level (0.1 min−1). The Lu12 DNAzyme was isolated using Lu3+ (Figure 11A). It is more active with the lighter Ln3+ showing a descending activity trend with the heavy ones.102 The Tm7 DNAzyme (Figure 11B) was isolated with Tm3+, which is active only with the seven heavy Ln3+.103 The Dy10a (Figure 11C) was isolated with Dy3+, and its highest activity is seen with the middle five Ln3+.104 A gel-based assay was performed for each DNAzyme with each Ln3+ showing the aforementioned activity trend (Figure 11D).102,104,270 These DNAzymes are all highly specific to Ln3+ (e.g., nearly inactive with nonlanthanides), but no DNAzyme has specificity for any particular Ln3+. A patternrecognition method was developed for discriminating the Ln3+ using a DNAzyme sensor array.270 Introducing Ln3+ to the DNAzyme chemistry has resulted in a few new reaction mechanisms. For example, the Tm7 and Dy10a cooperatively

bind three and two Ln3+ ions, respectively, and both have a very strong thio effect when the cleavage site was modified with a PS. We also obtained a DNAzyme that cleaves 2′-5′ linked RNA,271 whose metal selectivity is similar to the Lu12. A Gd3+ aptamer was recently reported (Figure 11E).272 Using a fluorescence-based assay, the optimized structure was characterized to have a Kd of 330 nM Gd3+, which was then converted into a sensor with a detection limit of 80 nM Gd3+. The sensor was also highly selective against many other divalent and trivalent metals, but it could not distinguish different Ln3+. Recently, DNA mimicking fluorescent nucleobases has been employed by Kool and co-workers to develop metal sensors. A sensor array was demonstrated to discriminate the 14 nonradioactive lanthanides with 100% identification accuracy.273 Yttrium (Y3+) is also a trivalent rare earth metal with a similar size to Ho3+, and its activity with the above lanthanidedependent DNAzymes is also similar to that of Ho3+.102−104,110 So far, no in vitro selection was carried out with Y3+. Adsorption of DNA by Y2O3 was studied, and the DNA phosphate backbone plays an important role in this adsorption reaction.274 Sc3+ is also a rare earth metal but it is much smaller than Y3+. This might be the reason that Sc3+ is inactive with all known Ln3+-dependent DNAzymes. 3.3.2. Uranium (UO22+). The actinide series also contains 15 metals. Among them, however, only thorium and uranium are accessible by most chemistry laboratories. The others are strongly radioactive and cannot stably exist. Uranium is widely used for making nuclear weapons and power generation, and detection of uranium as a nuclear waste is a practical analytical need. In water, UO22+ is the most stable form of uranium. UO22+ has a linear geometry with O being in the axial direction, and it coordinates to five H2O molecules located in the equatorial plane, complying with a bipyramidal like structure.275,276 Uranyl is a hard Lewis acid strongly yet reversibly coordinating to the DNA phosphate at low pH.277,278 With its strong DNA binding and strong electron scattering properties (due to high atomic number), uranyl salts have been used for staining DNA for electron microscopy.277 DNA bound UO22+, when irradiated with long wavelength UV light of 420 nm, induces single-stranded nicks,278,279 which was used for probing DNA structures.279−283 The Lu lab selected a DNAzyme named 39E (Figure 12) in 2007 with million-fold selectivity for UO22+ against other tested

Figure 12. Secondary structure of the UO2 2+ -specific 39E DNAzyme.101 The red nucleotides in the loop are conserved for catalysis.284

metals.101 The nucleotides important for catalysis resided in the bulge loop in red (Figure 12A),284 and the metal binding sites were later more accurately probed through a uranyl photocleavage study.285 Nucleotides at several positions are collectively responsible for uranyl binding, including the bulge 8284


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Figure 13. Structures and stability constants (determined at 25 °C, 0.1 M NaNO3) of (A) ribose monophosphate; (B) neutral nucleobases adenine, guanosine, and cytosine; (C) guanosine monophosphate; and (D) uridine monophosphate and phosphorothioate. The coordination sites of Pb2+ are highlighted in color with stability constants ≤1.0 as blue, between 1 and 3 as green, and ≥3 as red.

Figure 14. Secondary structures of (A) the leadzyme (a ribozyme), (B) the GR5 DNAzyme, and (C) the 17E DNAzyme. (D) A comparison of metal specificity of the GR5 and 8-17 DNAzymes. Adapted from ref 97 with permission from the Royal Society of Chemistry.

developing biosensors for Pb2+ has been a long-standing focus of research. The coordination of Pb2+ by nucleotides has been studied in detail since the 1960s attributable to the activity of Pb2+ in cleaving RNA. Pb2+ at ∼1 mM nonspecifically depolymerizes RNA at near-neutral pH.288 Crystallographic data showed that the catalytic Pb2+ coordinates with negatively charged oxygens of the phosphate along with the electron rich sites in selective nucleobases (e.g., N7 and O6 of guanine, N3 and O2 of cytosine, and O4 of uracil) near the cleavage site by acting like a general base in the form of Pb(OH)+.289 The pKa value of its metal bound water is close to neutral, making it ideal as a general base to assist activation of the 2′-OH nucleophile. At the individual nucleotide level, the stability of Pb2+ coordination to the phosphates of D-ribose 5-monophosphate is the highest (Figure 13A),290,291 followed by the neutral

loop between T23 and C25, G11 and T12 in the stem loop, and substrate strand close to the cleavage site between T2.4 and G3. A FRET-based method failed to find global folding of the 39E in the presence of UO22+, suggesting a “lock-and-key” mechanism for metal binding and catalysis.286 The 39E can detect UO22+ down to 45 pM.101 3.4. Post-Transition Metal Ions

Post-transition metals are located after the transition metals in the periodic table, including gallium, indium, thallium, tin, lead, and bismuth. Most of these metals are highly toxic, and their interactions with DNA are less explored except for lead. 3.4.1. Lead. Lead is a highly toxic heavy metal especially to children, causing serious developmental disorders and mental illness.287 Lead poisoning has been a serious concern due to historic reasons of lead in gasoline, pipes, and paint. Therefore, 8285


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Figure 15. (A) Chelation cages of K+-O and Pb2+-O in G4 DNA. Adapted with permission from ref 327. Copyright 2009 American Chemical Society. (B) Schematic drawing of the folded thrombin binding aptamer, G2T2G2TGTG2T2G2, with the placement of 2 K+ or 1 Pb2+ ion. Adapted with permission from ref 328. Copyright 2002 American Chemical Society.

Figure 16. (A) Scheme illustrating the PS2.M DNA folding into a G4 structure in the presence of Tl+.334 (B) Desulfurization of the PS substrate to the normal phosphodiester (PO) by Tl3+ allows its cleavage by the Tm7 DNAzyme in the presence of Er3+.336 The asterisk at the cleavage site denotes the PS linkage. Note that the Tm7 cannot cleave the PS substrate.

lized sensors.303 However, the 17E has relatively poor metal selectivity. In fact, the same 17E motif has been isolated in the presence of Zn2+, Cd2+, Ca2+, and Mg2+.78,227−230,304 The reason for the recurrence of 17E was explored by Li and coworkers, and they attributed it to its small size, high catalytic efficiency, and tolerance to mutation.230,305,306 Aside from Pb2+, Zn2+, and Cd2+ are the next most active metals.77,304 When it comes to real water samples, Ca2+ is a major concern especially for hard water. The recurrence of the 17E has also resulted in extensive biochemical studies.77,228,299,305−312 The Lu group suggested a distinct reaction mechanism of Pb2+ from other divalent metals.306 They found that Pb2+ activates the 17E via a lockand-key mechanism, while other metals need an adaptive-fitting process.313,314 CD spectroscopy showed a Z-DNA formation of the 17E in the presence of Zn2+ and Mg2+ but not with Pb2+.315 Contact photo-cross-linking experiments by Liu et al. indicate that Pb2+ triggers a local folding within the active site to activate the DNAzyme, while global folding is unnecessary.316 In 2010, the Lu group went back to examine the GR5 DNAzyme and found that it has remarkable specificity with Pb2+ (Figure 14D).97 It is essentially inactive with any other tested metal ions, even with 50 mM Mg2+. A comparison of the 17E and GR5 showed that they share the same crucial nucleotides for activity, suggesting that they may have a similar Pb2+ binding mechanism.299 We also performed a Pb2+-dependent selection and isolated many new DNAzymes that are highly specific for Pb2+.317 In addition to DNAzymes, G4 DNA was also exploited for Pb2+ detection. The strong and specific binding between Pb2+ and G4 structures has been noticed for a long time.68 Although many other metals such as Na+, K+, Tl+, Sr2+, and Ba2+ can also stabilize G4 DNA, Pb2+ is the most effective one. Typically,

nucleobases, N7 and C6(O) of guanosine, N3 and C2(O) of cytidine, and drops to extremely low for N1 and N7 of adenine, while no interaction is known with carbonyl oxygens of neutral uridine or thymidine with aqueous Pb2+ (Figure 13B).290−293 The increased stability of Pb2+ binding to GMP (as compared to the G base alone) reflects that the coordination of Pb2+ to the negatively charged phosphates facilitates its coordination to the N7 site (Figure 13C).290−292 Lead has a strong affinity toward sulfur, leading to the high binding stability constants with nucleoside phosphorothioates (Figure 13D).291,294,295 Studies on Pb2+-induced cleavage of tRNA have led to the discovery of the leadzyme (Figure 14A),296−298 a very small ribozyme that uses Pb2+. Breaker and Joyce in 1994 selected the first DNAzyme named GR5 using Pb2+ as the metal cofactor (Figure 14B).22 GR5 has a rate of ∼1 min−1 in this initial report, and it can reach >10 min −1 under optimized conditions.299 Back then, the DNAzyme field was focused on cleaving viral RNA for therapeutic applications instead of environmental applications. However, GR5 cannot cleave allRNA substrates (it cleaves only the RNA/DNA chimera), and it requires toxic Pb2+, making it impossible for in vivo applications. As such, little research was conducted on the GR5 for a long time.79 In 2000, a variant of the 8−17 DNAzyme named 17E (Figure 14C) was isolated in the presence of Zn2+,78 which was later found to be highly active with Pb2+ by Li and Lu, who for the first time converted the DNAzyme into a fluorescent biosensor for Pb2+.27 At neutral pH, the 17E can also reach >10 min−1 with just low micromolar Pb2+. Its detection limit for Pb2+ is typically below 10 nM and sometimes even below 1 nM.69,97 Since then, the 17E DNAzyme has served as a model system to establish a diverse range of signaling methods, such as fluorescence,27,300 electrochemistry,301 color,302 and immobi8286


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Figure 17. (A) Cu2+-specific DNA-ligating DNAzyme. Im denotes an imidazole group and P indicates a phosphate.349 (B) The Cu2+-specific DNAcleaving DNAzyme requiring ascorbate for oxidative DNA cleavage.115 In addition to the guanine pointed by the arrowhead, a few other nucleotides are also cleaved. (C) The Cu2+-dependent RNA-cleaving DNAzyme. The red asterisk indicates a PS RNA linkage.107 (D) A scheme showing visual and portable detection of Cu2+ based on a poly thymine functionalized hydrogel for templating the synthesis of fluorescent copper nanoparticles. Reprinted with permission from ref 365. Copyright 2014 American Chemical Society.

mM concentrations of K+ and Na+ are required to fold G4 DNAs, while low μM Pb2+ is sufficient. Although both K+ and Pb2+ are situated between two quartets of an octamer and coordinate with 8 carbonyl oxygens, Pb2+ exhibits smaller vertical separation of the G-quartets and a shorter G-quartet diameter than K+, reflecting a more compact and stable G8 cage (Figure 15A).318,319 For the same G4 DNA, different metal ions may induce different G4 structures, and this allows for metal detection.320,321 For example, while two K+ are required, only one Pb2+ is necessary to completely fold the thrombin aptamer shown in Figure 15B into two quartets stacked over each other. This remarkable conformation switching has been exploited to build multiple Pb2+ sensors and DNA logic gates.322−327 3.4.2. Thallium. Thallium is highly toxic, even more so than mercury. Thallium exists as both Tl+ and Tl3+ in nature, with Tl+ being the dominant one. The toxicity of Tl+ is generally attributed to its similarity to K+ in terms of ionic radii (Tl+ = 1.40 Å; K+ = 1.33 Å), bond lengths (2.4−2.7 Å), and both adopting irregular coordination geometries.329 As such, Tl+ can substitute K+ in biochemical processes and may block the normal functions of K+.330 Tl3+ is much more toxic than Tl+, which may relate to its strong thiophilicity. It is challenging to measure thallium with many common analytical instruments due to its high volatility. The interaction between Tl+ and DNA is well-documented. IR spectroscopy showed that each 1000 nucleotides bind 10− 30 Tl+ ions with a binding constant of 1.4 × 104 M−1.331 Taking advantage of Tl+ inhibiting the electroactivity of the N7 in guanine, it showed that nanomolar Tl+ does not cause a DNA conformational change or damage.332 Specific binding of Tl+ to G4 DNA was also known due to its similar size as K+.161−163,333 Recently, we screened a series of G-rich sequences for Tl+ binding and found the PS2.M DNA to be the best candidate for Tl+ sensing (Figure 16A).334 It appears that the kinetics of G4

DNA binding is slower for Tl+ than for K+, which might be due to the need to break the Tl−Cl bond in buffer. The interaction between Tl3+ and DNA was less explored. 3+ Tl was reported to oxidatively damage DNA even at nanomolar concentrations.335 We failed to select Tl3+-specific DNAzymes. It appears that, at neutral pH, Tl3+ interacts very weakly with DNA probably due to its hydrolysis. This is exemplified by the lack of inhibition on DNAzyme activity with even 1 mM Tl3+ (Hg2+ inhibits at 10 μM). While no new DNAzymes were obtained, the thiophilicity of Tl3+ still enables us to detect it using the lanthanide-dependent Tm7 DNAzyme (Figure 16B).336 The Tm7 cleaves only the normal phosphate (PO) RNA linkage, but it is completely inactive with the PSmodified substrate.103 Tl3+ desulfurizes the PS substrate to the normal PO substrate, allowing for its cleavage by the Tm7 in the presence of Er3+. 3.4.3. Aluminum. Al3+ is a neurotoxic agent, and its toxicity was suggested to be related to DNA binding.337,338 pHdependent interactions of Al3+ with DNA were characterized by isothermal titration calorimetry (ITC) and other techniques. The highest binding affinity was observed at pH 4.5. At higher pH, binding diminished, owing to hydrolysis and precipitation of Al3+.339 Because of its hard Lewis acid nature, Al3+ is believed to bind DNA mainly through the phosphate backbone,340 and its binding is about 4000-fold stronger than that of Mg2+.341 Such strong binding may be useful for Al3+ detection using DNA. While no Al3+-specific DNA sequences were reported yet, Ye and co-workers utilized its strong phosphate binding to construct a colorimetric probe for Al3+. In their design, ATP was adsorbed on the surface of gold/silver nanoparticles through base coordination, and the exposed phosphates chelated Al3+ to aggregate the nanoparticles resulting in a color change.342 We also performed in vitro DNAzyme selections for Al3+, together with some other group 3A metals such as Ga3+ and In3+.343 However, no active DNAzymes were 8287


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Figure 18. (A) Zn2+-dependent RNA-cleaving DNAzyme.371 The U in red denotes C5-imidazole-modified deoxyuridine. The secondary structures of the (B) 10MD5 and 9NL27 DNAzymes,378 (C) IR-3 DNAzyme,380 and (D) Zn-6m2 aptamer.92 The F in (D) indicates a fluorescein labeling.

by this DNAzyme is oxidative instead of hydrolytic. Other in vitro selections using Cu2+ were also carried out, but Cu2+ was not the only metal added in those studies (typically only part of a metal soup).352−354 Considering the thiophilicity of Cu2+, we introduced a PS modification at the substrate cleavage site and performed a selection in the presence of Cu2+.107 A few very small DNAzymes were isolated, and an example named PSCu10 is shown in Figure 17C. This DNAzyme is inactive in the presence of ascorbate, suggesting that it uses Cu2+ instead of Cu+. A catalytic beacon based on PSCu10 can detect down to 1.6 nM Cu2+ with excellent selectivity. In addition to DNAzymes, Cu2+ was also used in other DNArelated catalytic reactions. Li et al. discovered a unique coordination mode between Cu2+ and dsDNA containing tandem GC base pairs (i.e., GpG duplexes). The complex can significantly accelerate the oxidation of TMB in the presence of H2O2. In this regard, this complex is a Cu2+-dependent peroxidase mimic.355 Wang et al. designed a G-rich sequence that forms a G4 structure stabilized by Cu2+, which can catalyze the enantioselective Friedel−Crafts reaction.356 The coppercatalyzed click chemistry has also been used for Cu2+ sensing.357 For example, Ge et al. split a G-rich sequence into two pieces and modified the split fragments with an azide and an alkyne group, respectively. In the presence of ascorbate, Cu2+ was reduced to Cu+, which triggered the click reaction to link the two fragments to a full G4 structure, converting TMB into a colored product. Zhan et al. reported a ssDNA called Cu100 that undergoes specific folding in the presence of Cu2+ allowing a turn-off sensing of Cu2+ using SYBR Green I (SGI).358 However, the underlining binding mechanism is unclear. More recently, using the structure-switching aptamer selection method combined with emulsion PCR, Qu et al. reported a novel Cu2+ aptamer displaying a Kd of ∼50 μM with high specificity, but the structure of this aptamer has yet to be further characterized.95 Oomens and co-workers found that Cu+ can effectively stabilize the cytosine dimer to form the C-Cu+-C motif, reminiscent of the more well-known C-Ag+-C pair.359 The coordination of Cu2+ with DNA also facilitates the synthesis of fluorescent copper nanoparticles.360−362 Mokhir’s group initially found that only dsDNA could act as a template for such nanoparticles.363 Later, Qing et al. demonstrated that

obtained. At neutral pH, these metals are extensively hydrolyzed and thus may not assist the RNA cleavage reaction. 3.5. Transition Metals

Transition metals occupy a large portion of the periodic table and are of great analytical importance. Many transition metals can easily lose their bound water molecules for inner-sphere coordination, resulting in a high DNA binding affinity. Nonspecific interactions between DNA and transition metal ions have been studied by various techniques, which collectively indicate that metals prefer DNA bases to the phosphate backbone with binding affinity following Hg2+ > Cu2+ > Cd2+ > Zn2+ > Mn2+ > Ni2+, Co2+ > Fe2+.344 Efforts have been made to isolate their aptamers and DNAzymes. The rich spectroscopic properties of transition metals are also useful for fundamental understanding of metal binding by DNA.96 3.5.1. Copper. Copper is a commonly used heavy metal with moderate toxicity (toxic limit = 20 μM). At low concentrations, Cu2+ is an essential nutrient found in various enzymes. However, long-term exposure to high levels of Cu2+ can cause toxic effects.345 Cu2+ is a soft metal that strongly coordinates with the nucleobases in DNA. An early work explored the interaction of Cu2+ with both dsDNA and ssDNA, indicating that Cu2+ can intercalate into dsDNA with a Kd of 41 μM.346 This binding may even cause DNA lesions.347 Hackl et al. compared the coordination strength of different metal ions with DNA (Cu2+, Zn2+, Mn2+, Ca2+, and Mg2+). This study showed that Cu2+ is among the most effective ions to compact DNA, owing to its strong coordination with DNA bases facilitating DNA bending.348 Cuenoud and Szostak selected a DNA-ligating DNAzyme in the presence of Cu2+ (Figure 17A).349 This reaction however requires an unstable imidazole activated substrate. In addition it has a narrow dynamic range since Cu2+ becomes an inhibitor at concentrations higher than 10 μM. This DNAzyme is quite selective and is only active with Zn2+ (∼400-fold lower than that of Cu2+). It was used in a few cases for Cu2+ detection by measuring the ligation product.350 Taking advantage that Cu2+ is a redox active metal, Breaker and co-workers isolated a few DNA-cleaving DNAzymes using Cu2+ and ascorbate, and a representative one is shown in Figure 17B.114,115 A sensor was made to detect Cu2+ down to 35 nM using this DNAzyme.351 However, the need of unstable ascorbate also complicates this reaction. The DNA cleavage 8288


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Figure 19. (A) Example of Hg2+ binding aptamer designed based on the T-Hg2+-T interaction (structure shown in Figure 2G).65 Several other metallo-base-pairs stabilized by Hg2+ are also shown. (B) A cis-cleaving structure of the Hg2+-dependent RNA-cleaving DNAzyme containing modified nucleotides (in yellow): dAim, 8-histaminyl-dA; and dUaa, 5-aminoallyl-dU.397 (C) The active metals for the Ce13d DNAzyme when using the PS-modified substrate. Reprinted with permission from ref 69. Copyright 2014 American Chemical Society. (D) Direct cleavage of a PS-RNA by Hg2+, and three types of products are generated.399

poly thymine ssDNA can also be used.364 The particle size and optical properties can be fine-tuned by simply altering the number of thymine bases in the ssDNA. With this knowledge, they further developed a poly(thymine)-caged and microwellprinted hydrogel for visual detection of Cu2+ (Figure 17D).365 3.5.2. Zinc. Zn2+ plays multiple biological roles including being a cofactor in metalloproteins.366 Up to 10% of human coding proteins contain at least one Zn2+ binding motif.367 A high concentration of Zn2+ however can be toxic. Given its importance, detection of Zn2+ is a long-standing task in analytic chemistry.14 Zn2+ binds to both hard and soft ligands including nitrogen, oxygen, sulfur, and halogen with coordination numbers of 4−6. Due to an extremely low energy penalty of changing coordination number of hydrated Zn2+ (e.g., < 0.4 kcal/mol for Zn(H2O)62+ to Zn(H2O)52+·H2O/Zn(H2O)42+·2H2O), Zn2+ has more of a catalytic role rather than structural role in aqueous enzyme systems.368 Zn2+ binding to DNA is weaker than Cu2+, Cd2+, and Pb2+ but stronger than Mn2+, Mg2+, and Ca2+.17,369,370 Zn2+ is a strong Lewis acid. With the assumption that Lewis acidity might be important for RNA cleavage, it has been utilized in many DNAzyme selections. Lu and co-workers used Zn2+ to isolate RNA-cleaving DNAzymes but produced the 17E.78 Joyce and co-workers used a library containing imidazole-modified uridine and isolated a new Zn2+-dependent DNAzyme (Figure 18A).371 However, no metal selectivity test was carried out, and little follow-up work was performed on this DNAzyme probably due to its poor commercial availability. In vitro selection of Zn2+-dependent DNAzymes at high temperature was also performed,372 and the resulting DNAzymes exhibited Zn2+-specific RNA cleavage activity. The above Cu2+dependent DNA-ligating enzyme works with Zn2+ as well.349 A series of selections were performed by the Silverman group to isolate RNA-ligating DNAzymes that use multiple metals including Zn2+.373,374 Recently, they expanded their selection to

DNA cleavage, and a representative DNAzyme called 10MD5 was found (Figure 18B), which required both Zn2+ and Mn2+.265,375−377 A follow-up study identified a Zn2+-specific mutant named 9NL27.378 The 9NL27 differs from the original 10MD5 only by five nucleotides (Figure 18B, the nucleotides in red). A biosensor was designed to detect Zn2+ using the 9NL27, and a PCR process was incorporated to improve the detection limit.379 Later, Breaker and co-workers selected DNA-cleaving DNAzymes with Zn2+ alone using a circular library. A very small DNAzyme called I-R3 was obtained with a rate of 1 min−1 in the presence of 2 mM Zn2+ (Figure 18C).380 This DNAzyme was then made into a biosensor that reported a detection limit of 1 nM Zn2+.381 The search for Zn2+ aptamers has been attempted a few times. The Yarus group first explored RNA aptamers by immobilizing Zn2+ on a column. The isolated sequences have a Kd of 100−400 μM Zn2+.21,382 In 2000, Sugimoto and coworkers reported a few Zn2+-dependent RNA aptamers that bind with the HIV-1 Tat protein. Although the aptamers were isolated for the protein target, Zn2+ was essential for the binding, suggesting a Zn2+ binding motif in the aptamers.383 Using the structure-switching selection method (Figure 3),91 the Ellington group selected a few signaling DNA aptamers for Zn2+. A representative one called Zn-6m2 (Figure 18D) detected Zn2+ down to 5 μM, but it was strongly interfered by Cd2+.92 Many selections and activity assays were carried out using a relatively high Zn2+ concentration (∼1 mM) at neutral pH, under which Zn2+ might hydrolyze and precipitate. Zn(OH)2 or ZnO precipitations are positively charged to strongly adsorb DNA and inhibit DNA activity.384 Indeed, we noticed that some DNAzymes were quickly inhibited when Zn2+ concentration was beyond 1−2 mM at neutral pH.384 3.5.3. Mercury. Mercury is a highly toxic metal. The literature on sensing mercury using DNA has grown drastically since 2004 thanks to the thymine binding scheme in Figure 2G. 8289


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desulfurized, Figure 19D).399 The overall cleavage efficiency can be increased by incorporating multiple PS RNA sites, allowing this direct cleavage reaction to be useful for Hg2+ detection. An advantage of such a reaction is a fast response time; cleavage is finished within 2 min. In addition, the reaction is tolerant to different buffer conditions, and the existence of Cl− has little effect on the reaction. It needs to be noted that the T-Hg2+-T interaction is strongly inhibited by Cl− and other chelators.400 3.5.4. Cadmium. Cd2+ is a toxic heavy metal and a known carcinogen.3 Cd2+ is widely used in many industrial processes and people can get exposed even by smoking cigarettes. Cd2+ is a soft metal and shows a range of coordination numbers from 2 to 8. The coordination stability of Cd2+ increases with the softness of donor atoms (S ≫ N > O). Cd2+ is tightly coordinated by the nucleobases.17,369,401 A few attempts of isolating Cd2+-specific DNAzymes were made. Kasprowicz et al. performed a Cd2+-dependent selection, but it resulted in variants of the 8-17 DNAzyme.304 Mutating the resulting DNAzymes can slightly shift its activity with Cd2+, Zn2+, and Mn2+. Although certain sequences gave the highest cleavage with Cd2+ (Pb2+ was not tested, Figure 20A), the

The specific interaction between thymine-rich DNA and Hg2+ was noticed since the 1950s from the UV−vis studies.385 It has caught the attention of analytical chemists with the paper by Ono and Togashi demonstrating that a thymine-rich molecular beacon was specifically quenched by Hg2+.65 This particular sequence (Figure 19A) has become a popular mercury aptamer used by many groups to rationally design Hg2+ sensors, although other thymine-rich DNAs can also be used. This discovery is very powerful for its simplicity. Multiple Hg2+ binding sites can also be engineered for cooperative binding. A variety of fundamental studies were performed using techniques such as NMR,59,386 UV−vis spectroscopy,59 and X-ray crystallography.84 Isothermal titration calorimetry (ITC) revealed a Kd of 1 μM Hg2+ toward each single T:T mismatch.387 The U-Hg2+-U binding in RNA was also reported and characterized by 1H NMR spectroscopy.388 Other metallobase-pairs stabilized by Hg2+ were also demonstrated and are shown in Figure 19A, including T-Hg2+-C and A-Hg2+-T.66,389 The importance of such a T-Hg2+-T DNA hybrid in the field can be reflected by the wide range applications derived from it, such as metal detection,65,390 Hg2+ trapping,391 single nucleotide polymorphisms (SNPs) detection,392 and DNA nanomachines.393 These applications in turn stimulate further fundamental studies on this metallo-base-pair. It is found that the Hg−N3 bond in T-Hg2+-T was found to be less covalent but more ionic.394 This ionic nature enables Hg2+ to act as an electron acceptor, which can quench fluorescence through electron transfer from the labeled fluorophore to the T-Hg2+-T pair.395 A three-dimensional (3D) structure of tandem T-Hg2+T pairs was determined by NMR spectroscopy, indicating that the DNA was in a B-form double helix and that the Hg2+ ion was completely shielded from the solvent water.396 The Hg2+ in the structure is dehydrated, which would explain the positive reaction entropy for T-Hg2+-T formation.396 X-ray crystal structure has showed that the distance between N3 and Hg2+ is 2.0 Å, indicating that N3 has been deprotonated.84 More detailed structural and chemical prescriptions for such T-Hg2+T metallo-base-pair can be found in specific reviews.66,389 Recently, a new Hg2+ aptamer was identified by selection, which claimed to have an even higher Hg2+ binding affinity than thymine-rich DNA.95 Using Hg2+ as a cofactor for selecting RNA-cleaving DNAzymes has been a challenging task. No effective unmodified DNAzymes using Hg2+ were reported. The Perrin group succeeded by incorporating two modified DNA bases (Figure 19B).397 However, most researchers do not have access to such modified DNAzymes, and this DNAzyme has not been tested as a biosensor. For the RNA cleavage reaction shown in Figure 4C, an important requirement is metal binding to the scissile phosphate. However, mercury as a strongly thiophilic metal has very low affinity to the phosphate, which might explain its chemical limitation in RNA cleavage. The problem can be solved by using PS modification.69,398 For example, by using a PS substrate, the Ce13d DNAzyme was switched from being lanthanide-specific to being reactive with thiophilic metals such as Hg2+, Pb2+, and Cd2+ and, to lesser extent, Cu2+ (Figure 19C).69 In fact, the thiophilicity of Hg2+ is so strong that, even without a DNAzyme, a PS containing RNA alone can be cleaved by Hg2+, although the efficiency of this reaction is low. Only ∼16% of PS-RNA molecules undergo cleavage, and the rest are desulfurized to the normal RNA with a phosphodiester linkage or isomerized to form 2′-5′-linked RNA (still

Figure 20. (A) Metal-dependent activity of the cis-cleaving 8-17 and a few mutants. The mutated nucleotides are numbered and highlighted in purple. In the table below, the relative maximal cleavage yields are compared with the wild-type DNAzyme (upper panel) after 30 min incubation in the presence of 50 μM metal.304 (B) The Cd16 DNAzyme with the PS-modified substrate.106 The red asterisk at the cleavage site indicates the PS-modified RNA linkage. The secondary structures of the (C) Cd494 and (D) Cd-2-2 aptamers for Cd2+.93

difference was quite moderate, and it is unlikely to be useful for Cd2+ detection. The Li group used Cd2+ in a series of signaling DNAzyme selections.402 Their library contained a fluorophore and a quencher right next to the cleavage site. Because of this special design, they avoided the 8-17 motif. In addition, the selected DNAzymes can be directly used as metal sensors. However, none of the obtained DNAzymes were very specific for Cd2+. Similar to Hg2+, we reason that the difficulty of obtaining Cd2+-dependent DNAzymes might be its thiophilicity. There8290


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surplus causes adverse health effects.412,413 The U.S. EPA has set the safety limit of Mn2+ concentration in drinking water to be 5.5 μM (lifetime exposure).414 Sub-mM Mn2+ induces the transition of B-DNA to ZDNA.415 A few Mn2+ responsive riboswitches have been discovered, which can sense Mn2+ at the low μM range with a high selectivity for Mn2+ over Mg2+ and Ca2+.416,417 Mn2+ is a moderately soft metal, and it can bind to both oxygen and sulfur ligands. For this reason, it is a widely used probe for metal binding sites in ribozymes when PS modifications are made.81,418 In addition, due to its paramagnetic nature, Mn2+ has also been used as an electron paramagnetic resonance (EPR) probe for nucleic acids.419,420 The search of DNA aptamers for Mn2+ binding was less explored. In vitro selection of DNAzymes using Mn2+ alone was reported by the Li group, but they still yielded the 8-17 motif.230 Li and co-workers also selected a series of DNAzymes using metal mixtures (Cd2+, Mn2+, Ni2+, and Mg2+) at various pH levels.402 In this case, a fluorophore and a quencher were respectively labeled flanking the cleavage site. The one obtained at pH 7 (called pH7DZ1) displayed a relative high specificity for Mn2+. A selection using a metal mixture was also performed by Zivarts et al. to identify allosteric hammerhead ribozymes.421 The obtained ribozymes are similarly activated by Mn2+, Fe2+, Co2+, Ni2+, Zn2+, and Cd2+. Mn2+ was also used together with other metals for selecting DNAzymes with a diverse range of activities beyond RNA cleavage.354,422−427 3.5.7. Nickel. Nickel can exist in many different oxidation states with +2 being the most common under ambient conditions. Nickel is believed to be an essential element in biology, while its biological functions have yet to be fully understood. The toxicity of nickel is moderate, and the most commonly suffered health effect of nickel exposure is an allergic reaction such as contact dermatitis.428 The nickel level in drinking water was limited to 1.7 μM by the U.S. EPA. The interest to find Ni2+ binding aptamers has dated back to 1997, when an RNA aptamer was isolated by immobilizing Ni2+ on a resin.90 Although the aptamer preferentially binds Ni2+ over some other transition metals with a high affinity (Kd = 1 μM), Co2+ and Ni2+ were virtually indistinguishable by this aptamer. DNAzyme selection with Ni2+ was also performed in combination with other ions, but the resulting DNAzyme showed low specificity.402 The interaction between Ni2+ and double-stranded DNA at slightly basic pH was studied by Lee and co-workers. They proposed that the imino proton in each base-pair of the duplex is replaced by Ni2+, forming the so-called M-DNA.429 Zn2+ and Co2+ under alkaline conditions can also cause a similar binding. The concept of M-DNA has been quite attractive, and its electric conductivity has been measured.430 However, others have questioned about the model of M-DNA and suggested that observations might simply be due to aggregation of DNA.431 3.5.8. Iron. Iron is the most abundant transition metal in biology, and it plays crucial roles for carrying oxygen and in forming metalloenzymes. Iron exists mainly in Fe2+ and Fe3+. Fe2+ is unstable under ambient conditions and is easily oxidized to Fe3+, while Fe3+ has a poor solubility in aqueous solution. At the physiological pH of 7.4, its solubility is only about 10−18 M as free ions, while the rest are hydrolyzed.432,433 Distinguishing between Fe2+ and Fe3+ has attracted considerable research interest for their environmental and biological importance. Their differences in electronic structures and redox potentials

fore, introducing a PS modification at the cleavage site might help. With a PS modification, a DNAzyme named Cd16 was isolated (Figure 20B).106 It cleaves the PS substrate with rate of 0.12 min−1 in the presence of 10 μM Cd2+ at pH 6.0. Moreover, it prefers the Rp diastereomer, and the rate is ∼100-fold faster than that with the Sp substrate. Although Hg2+ also induces ∼8% of cleavage within 20 s (due to direct PS reaction rather than enzymatic cleavage), its response can be filtered out by plotting the fluorescence kinetics, by which an excellent metal specificity was achieved. Using unmodified DNA libraries, a few aptamer selections were performed in the presence of Cd2+. For example, Zhou and co-workers used the method in Figure 3 and obtained a series of Cd2+-binding aptamers. The most representative one called Cd-4 (Figure 20C) was further characterized by CD spectroscopy.94 More recently, Wang et al. performed the same SELEX experiment and reported another Cd2+-dependent aptamer, named Cd-2-2 (Figure 20D).93 It is interesting to note that these two aptamers had little sequence similarity. Both aptamers were converted to sensors for Cd2+. Their selectivity and sensitivity, however, could not rival the sensor based on the best DNAzymes.106 3.5.5. Cobalt. For the next six metals (cobalt, manganese, nickel, iron, molybdenum, and tungsten), no DNA or RNA based high quality sensors were demonstrated yet, and we briefly review their binding to nucleic acids and selection efforts to set the stage for future research on this front. Cobalt is an essential element in biology and a well-known example is vitamin B12, which is also called cobalamin.403 In addition, many cobalt-containing enzymes have been isolated and characterized.404 Wrzesinski et al. selected RNA aptamers for Co2+ by immobilizing Co2+ on a resin. Two RNA aptamers were isolated with a binding affinity of low mM Co2+,405 which was quite weak and approached nonspecific Co2+ binding. Co2+ at mM concentrations can activate the HDV406 and glmS ribozymes.407 By inserting a random sequence in the stem II of the hammerhead ribozyme for in vitro selection, Breaker and co-workers isolated an allosteric ribozyme containing a Co2+ binding domain.408 Such an allosteric design can sense Co2+ down to 1 μM with a Kd of 100 μM. However, it still suffers from poor specificity. Later, they discovered a class of riboswitches that could selectively and tightly bind to multiple Co2+ ions cooperatively with Kd down to ∼6.5 μM Co2+.34 Only Ni2+ also showed a positive binding with an affinity similar to that of Co2+, and thus they are called “NiCo” riboswitches. DNAzyme selection using Co2+ was reported by Lu and coworkers.108 Negative selections were introduced to improve metal specificity. To do so, the library was incubated with a mixture of competing metals, and the inactive sequences were collected for the positive Co2+ selection. Without negative selections, the resulting library was more active with Pb2+ and Zn2+. After negative selections, its Co2+-dependent activity became higher than that with Zn2+ and Pb2+. Further characterization unveiled the importance of the peripheral nucleotides at the substrate binding arms for metal selectivity.409 However, the selectivity of these DNAzymes still cannot meet the analytical need for Co2+ detection. 3.5.6. Manganese. Manganese is an essential micronutrient. A large number of enzymes require Mn2+ as cofactor for various functions (such as transferases, decarboxylases, and hydrolases).410,411 The normal Mn2+ level is about 70−270 nM in blood and only 7−15 nM in the fluid portion of serum. The Mn2+ level in biology is tightly controlled, as its deficiency or 8291


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Figure 21. Chemical structures of (A) Mo-co442 and (B) W-co.439 The metal components are highlighted in red.

Figure 22. (A) Phosphate concentration-dependent cleavage of the Ce13d DNAzyme with 100 μM Ce3+, Y3+, Pb2+, or Cr3+ after 1 h incubation. (B) Metal specificity test of the Ce13d-based sensor with different metal ions (4 and 20 μM) in the presence of 0.8 mM inorganic phosphate (1 h incubation). Inset: A scheme showing Cr3+ detection using the Ce13d. (A) and (B) were reprinted with permission from ref 447. Copyright 2016 John Wiley & Sons, Inc. (C) Fraction of bound DNA as a function of Cr3+ concentration after 24 h incubation. Reprinted with permission from ref 449. Copyright 2016 American Chemical Society.

have enabled a fluorescent method (based on QDs) for kinetic discrimination of Fe2+ and Fe3+.434 In contrast to our current oxidative environment, the early earth is anoxic and abundant in Fe2+. The coordination chemistry of Fe2+ is very similar to that of Mg2+ in many aspects. Quantum mechanical calculations indicate that Mg2+ and Fe2+ are coordinated by RNA phosphate with almost identical geometry, displaying quite similar metal−oxygen distances and angles.435 It was thus proposed that Fe2+ was an important metalloenzyme cofactor at that time, akin to Mg2+ for the modern life. To support this hypothesis, Williams and co-workers tested the activity of ribozymes in the presence of Fe2+, and found that Fe2+ was indeed an excellent Mg2+ mimic. Fe2+ readily replaces Mg2+ in RNA folding and catalysis and even in PCR.435,436 In fact, Fe2+ can achieve even better activity than Mg2+, which is likely due to a more favorable Fe2+− phosphate interaction, enabling a better activation for phosphoryl transfer. Ditzler and co-workers performed two parallel ribozyme selections using Mg2+ and Fe2+, respectively.437 Self-cleaving ribozymes have been successfully isolated in each case,

supporting that Fe2+ is indeed as good as Mg2+ for RNA catalysis. It should be noted that, for Fe2+ related work, experiments need to be performed in a glovebox under continuously bubbling argon to avoid oxidation of Fe2+. 3.5.9. Molybdenum and Tungsten. Molybdenum (Mo) and tungsten (W) are mainly in the hexavalent/tetravalent oxidation states. Their biological importance has already been identified.438 Mo is the only second-row transition metal that is essential to most living organisms. The species that do not require Mo need W, the heaviest element known to be of biological necessity.439 These two metals have been found in the active sites of enzymes exclusively in their coordination complexes (called molybdenum cofactor (Mo-co) and tungsten cofactor (W-co), respectively, Figure 21).440,441 Riboswitches for Mo-co have been discovered. The riboswitch recognizes and binds Mo-co and then undergoes a conformational change to shut down translation of the related mRNA.35 Although the binding mechanism has not been elucidated due to the liability of isolated Mo-co, an exceptional specificity has been demonstrated by riboswitch’s ability to discriminate Mo-co from W-co, two very similar cofactors.35 8292


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Figure 23. (A) Scheme of Ag+-mediated formation of a hairpin structure of a C-rich DNA for Ag+ detection.67 (B) Poly-C templated synthesis of AgNCs using NaBH4 as reduction agent, and further addition of free Ag+ induces dimerization of AgNCs, shifting the fluorescence from red to green.467 (C) The coordination sites on guanine for Ag+.54 (D) Ag+ coordination with two guanines forming a dimer.473 (E) The secondary structure of the Ag10c DNAzyme.119

22C). In a sense, Cr3+ is similar to Pt2+ since both can bind DNA very strongly with slow binding and dissociation kinetics.86

Moreover, some variants of the Mo-co riboswitch shift their selectivity from Mo-co to W-co.35 Therefore, the metal is an essential element for riboswitch binding. No work was reported on selecting aptamers or DNAzymes for these two metals. 3.5.10. Chromium. Chromium can exist in nine different oxidation states with the most common ones being Cr3+ and Cr(VI). Low concentrations of Cr3+ are considered to be a nutrient, but it is toxic at high concentrations. Cr(VI) is a strong carcinogen, and its toxicity is likely due to in situ reduction to Cr3+ and subsequent causing DNA lesions and cleavage.443 Cr3+ can bind to the N7 of guanine and also be chelated by a nearby phosphate.444 From the inorganic chemistry standpoint, Cr3+ has a d3 electronic configuration, and it prefers an octahedral ligand field, leading to a very slow ligand exchange rate of ∼9 × 10−3 h−1.86 As a result, Cr3+ can strongly coordinate with DNA, forming highly stable metalated DNA lesions and causing genotoxicity.445,446 We performed in vitro selection of DNAzymes using Cr3+. The main active sequences were however the previously reported lanthanide-dependent Ce13d DNAzyme. The Ce13d is about 150-fold more active with lanthanides (such as Ce3+) compared to the same concentration of Cr3+.447 The role of Ce3+ for the Ce13d is to stabilize the negatively charged transition state at the scissile phosphate. It seems that Cr3+ can also fulfill this role, albeit with a much lower efficiency. Interestingly, inorganic phosphate can fully mask the activity of lanthanide ions, Pb2+ and Y3+, while the Cr3+ activity is largely unaffected below 5 mM phosphate (Figure 22A). After masking, the Ce13d DNAzyme was used for highly sensitive and selective Cr3+ sensing (Figure 22B).447 Cr(VI) is an anion, and it has almost no interaction with DNA in buffer.448 By reducing Cr(VI) to Cr3+ using NaBH4, it can then be detected using the Ce13d. With a careful study, we noticed two types of binding between Cr3+ and DNA.449 Cr3+ binding to the DNA phosphate backbone is electrostatic and reversible. In addition, Cr3+ can strongly and irreversibly bind to DNA nucleobases and even cross-link DNA, which can survive harsh denaturing conditions with a high concentration of EDTA. Further understanding was achieved by studying the Cr3+ binding with DNA homopolymers, showing the apparent binding affinity to Cr3+ follows the order of G > C > A ≈ T (Figure

3.6. Noble Metals

Noble metals such as silver, gold, platinum, and palladium are also transition metals. We discuss them separately for their special properties. First, such noble metals are not endogenous elements in living organisms, and they do not actively participate in native biological processes. In addition, these metals are resistant to oxidation, and they can form stable metal nanoparticles for DNA adsorption.450−453 In this review, we only discuss their metal ions. Finally, these metals interact strongly with DNA bases but weakly with the DNA phosphate backbone. Some of these metals are of medicinal importance. For example, cisplatin is one of the most successful anticancer drugs, and its mechanism of action is believed to be related to irreversible DNA binding.58 Gold compounds have been used for treating arthritis, while silver is useful for antimicrobial purposes.454,455 Since these valuable metals are rare, increasing efforts have been devoted to develop their sensors.450,451 3.6.1. Silver. Cytosine-rich DNAs have been commonly used for Ag+ detection.67 Similar to the specific interaction between Hg2+ and thymine, Ag+ can selectively stabilize cytosine−cytosine mismatches (Figure 2F). In a 21-mer DNA duplex containing a single C−C mismatch, Ag+ raised the Tm by 8 °C, while other divalent metals failed to promote such a stabilization. ITC analysis confirmed the 1:1 binding molar ratio between Ag+ and C−C mismatch, showing a binding constant of 1 μM Ag+, which is significantly stronger than nonspecific metal−DNA interactions.456 Based on this C-Ag+-C binding, a representative sensor design is shown in Figure 23A, in which the C-rich sequence folds into a hairpin in the presence of Ag+, leading to quenched fluorescence. Besides the C-Ag+-C base pair, a few other pairs such as T-C, C-A, and some artificial nucleobases can also be stabilized by Ag+, with their thermal stabilities being comparable to that of C-Ag+-C.457−459 Among them, one such cytosine analog, pyrrolo-cytosine (pC), was further used for Ag+ detection, taking advantage of its intrinsic conformational-sensitive fluorescence.460 Free pC emits strongly in ssDNA, while its 8293


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Figure 24. (A) Proposed scheme of coordination between Au3+ and adenine.483 (B) A scheme of adenine/Au3+ coordination mediated assembly of coordination polymers.483 (C) Schematic of Au3+-rigidified poly-A DNA, resulting in a decreased methylene blue current signal for Au3+ detection. MB denotes for methylene blue. Reprinted with permission from ref 493. Copyright 2016 American Chemical Society.

∼0.4 min−1 with just 10 μM Ag+ and excellent selectivity for Ag+. Using a catalytic beacon design, a detection limit of 24 nM Ag+ was achieved. Further DMS footprinting studies indicate the presence of a silver aptamer binding pocket explaining its specificity for Ag+.480 3.6.2. Gold. Gold exists mostly in 0, +1, and +3 oxidation states, which strongly affects the properties of gold.481 For example, Au+ appears to be a soft metal, preferring sulfur over oxygen. Au3+, in contrast, is a hard metal and prefers binding to oxygen. The interaction between gold and DNA has been extensively studied using gold surfaces of Au0. For example, poly-A DNA can be tightly adsorbed by a gold surface, while poly-T DNA is adsorbed less tightly.482 Since Au0 is strongly thiophilic, it interacts mainly with the DNA bases with little binding to the phosphate backbone. Au3+ forms coordination polymers with nucleobases, nucleosides, and nucleotides.18 For example, Wei and Wang produced nanoparticles by mixing adenine and Au3+, and they proposed a coordination mode on adenine as shown in Figure 24A.483 The nanoparticles were made of aggregated small 2−3 nanoclusters (Figure 24B). We also used adenosine, AMP, and ATP as ligands and studied their Au3+ coordination complex under reducing conditions, resulting in luminescent complexes and particles.484,485 The strong coordination between DNA and Au3+ is also evidenced by DNA templated synthesis of AuNPs and gold nanoclusters (AuNCs) using Au3+ as a precursor.486−488 For example, Xu and Wang and co-workers prepared AuNPs by reducing HAuCl4 with citrate in the presence of DNA.489 The as-prepared AuNPs was in situ capped with DNA, and the complexes were highly tolerant to salt-induced aggregation. In addition, the functionality of DNA was maintained on the AuNPs surface, which can be further used for DNA-directed AuNPs assembly. We reported the use of poly-A (at neutral pH) and poly-C DNA (at low pH) to prepare and stabilize the fluorescent AuNCs.490 By varying the type of reducing agent and DNA length, the quantum yields were improved and different emission colors were obtained.491

fluorescence strongly diminishes after forming a pC-Ag+-C base pair. Since 2004,461 DNA-templated synthesis of fluorescent silver nanoclusters (AgNCs) has been carried out.462−464 C-rich DNA is the most frequently used template for AgNCs, but other DNA sequences such as poly-G can also be used.465,466 In one study, Lee et al. proposed that poly-C templated AgNCs can form a dimer upon addition of free Ag+ ion. Such dimerization of AgNCs shifts the fluorescence from red to green, allowing for Ag+ quantification (Figure 23B).467 Cytosine-rich DNA can also form the i-motif structure under acidic conditions.468 An i-motif is made of two parallel-stranded DNA duplexes hydrogen bonded together through C−C+ base pairs.469 Recently, Waller and co-workers found that i-motifs can be stabilized by Ag+ under physiological pH and unfolded by adding cysteine to remove Ag+.470 Inspired by this, Kang et al. designed a label-free i-motif DNA sensor for Ag+ detection using thiazole orange as a fluorescent stain.471 Silver also strongly coordinates with guanine.472 The N7 and O6 of guanine are mainly responsible for silver binding (Figure 23C).54 Intermolecular guanine coordination results in a stable dimer structure (Figure 23D), and Loo et al. reported GMP assembly and aggregation with this type of coordination.473 Since the silver binding sites in guanine are also important for G4 formation, Ag+ can disrupt the G4 structure, which was harnessed for Ag+ detection.474−477 The coordination between silver and adenine was employed for silver detection as well. Tan et al. engineered AMP/Tb3+ coordination polymers, which initially emitted luminescence very weakly. In the presence of Ag+, however, the same sample became strongly luminescent due to enhanced energy transfer from adenine to Tb3+.478 Recently, we studied the interaction between Ag+ and different DNA homopolymers by sensitized Tb3+ luminescence and found that the emissions from all of the sequences were enhanced by Ag+.479 Searching for silver-dependent DNAzymes was not pursued until very recently. We discovered a DNAzyme for Ag+ named Ag10c by in vitro selection (Figure 23E).119 It has a rate of 8294


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Figure 25. (A) Structures of various platinum containing compounds to be detected, and a scheme illustrating fluorescence quench by the adsorption of these compounds and subsequent fluorescence recovery by DNA-induced platinum desorption. Reprinted with permission from ref 498. Copyright 2014 Elsevier Science. (B) A scheme showing the detection of Pt2+ by Pt2+-mediated G-Pt2+-G base coordination to form an active SA aptamer. Reprinted with permission from ref 499. Copyright 2015 American Chemical Society.

Figure 26. (A) General secondary structure of RNA-cleaving DNAzymes. In presence of a metal cofactor, the substrate is cleaved into two fragments, and the cleaved fragments release from the enzyme strand. (B) A classical design of catalytic beacon. F and Q denote for fluorophore and quencher labels, respectively.

A few fluorescent and colorimetric sensors for Au3+ have been published,450,481 but DNA-based sensing has been less explored. No Au3+-activated DNAzymes were reported so far. Kwon et al. incorporated Au3+ into dsDNA to form DNA−Au3+ complexes. After Au3+ intercalation, the electrical conductance of DNA was altered depending on the Au3+ content.492 The strong coordination between adenine and Au3+ was used by Lai and co-workers recently. They reported an electrochemical sensor for Au3+ using just a poly-A DNA.493 The DNA was modified with methylene blue on one end, and the other end was immobilized on a gold disk electrode. The ssDNA attached to the gold surface is flexible, allowing effective electron transfer from methylene blue to the electrode. Addition of Au3+ rigidifies the DNA through interstrand DNA coordination and thus restricts the methylene blue from accessing the electrode, resulting in a decrease in the current signal (Figure 24C). 3.6.3. Platinum. The interaction between Pt and DNA is a classic topic in bioinorganic chemistry for the anticancer activity of cisplatin.494 It is believed that the ultimate target of cisplatin is DNA, and guanine is the preferred base for cisplatin binding.494,495 A key property of Pt is its very slow ligand exchange rate.87 Thus, the interaction between Pt and many ligands are kinetically controlled, resulting in very stable binding. For example, nitrogen and sulfur based ligands are very difficult to dissociate once they coordinate with Pt. Even relatively weak oxygen-based ligands, such as citrate, can effectively impede the association between cisplatin and DNA.496 Since Pt binds DNA tightly, this binding gives rise to metalated DNA. Platinated DNA carries a positive charge at each binding site, and it has been used to increase the binding affinity between DNA and graphene oxide (GO).497 However, such a slow ligand exchange rate also poses significant challenges for its detection, as the reaction may take a long and unpredictable amount of time depending on its original coordination state.

The strong and stable interaction between Pt and DNA has allowed for platinum drug detection using DNA as competitive ligand. In one study, He and co-workers adsorbed various platinum containing drugs on quantum dots (QDs), resulting in quenched QD fluorescence attributable to photoinduced electron transfer (Figure 25A). The addition of DNA dissociated platinum from the QDs’ surface, resulting in fluorescence recovery.498 This system was employed to detect cisplatin. By varying DNA sequence and length and using ssDNA/dsDNA, the interaction between DNA and platinum drugs was also studied. Thanks to the favorable coordination between Pt and guanine, an aptasensor was developed for direct sensing of Pt2+.499 In this system, a streptavidin (SA) aptamer labeled with FITC was folded into its binding structure in the presence of Pt (Figure 25B). Specifically, two base pairs in the aptamer sequence were replaced by G-G mismatches. Only in the presence of Pt2+ can the aptasensor be activated through Pt2+mediated formation of G-Pt2+-G base pairs. Then it can bind to SA modified magnetic beads for aptamer separation. Finally, anti-FITC-HRP (horseradish peroxidase) was added, and the bound HRP was used to catalyze the reaction between luminal and H2O2 for chemiluminescence signal. 3.6.4. Palladium. Pd usually coexists with Pt, and they are quite similar in chemical properties. Over a decade ago, an RNA aptamer was claimed to bind Pt nanoparticles.500 This work, however, was recently retracted due to technical artifacts and other reasons. More recently, a Pd2+ aptamer was isolated using a novel GO-adsorbed nanoparticles method.501 While so far no DNA based biosensors for palladium were reported, DNA was used to template the synthesis of various Pd nanostructures.502−506

4. SIGNALING METHODS The previous section mainly focuses on individual DNA sequences that can recognize each metal with little discussion 8295


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Figure 27. (A) Metal-induced disassembly of DNAzyme-linked AuNPs resulting in a blue-to-red color change upon cleavage.302 (B) DNAzyme cleavage triggers substrate release and an enzyme conformational change to switch on an electrochemical signal.301 (C) DNAzyme cleavage releases AuNPs from gold coated surface changing the SERS signal.526 (D) The cleavage reaction releases an invertase enzyme from the DNAzyme into solution. The DNA−invertase conjugate can hydrolyze sucrose to glucose, which is quantified by a glucose meter.527

the cleaved fluorophore-bearing fragment.509 Both the fluorophore and quencher can be labeled on the two ends of the substrate strand, and the two ends were also brought to proximity to form a molecular beacon structure.99 It is also possible to label a fluorophore/quencher right next to the cleavage site to maximize the signal change with a fast signaling kinetics.109,510 Many variations of this method have been developed by introducing other materials.511 For example, the fluorophore was replaced by QDs to further enhance sensitivity,512 and the quencher was replaced by another fluorophore for ratiometric signaling based on FRET.513 Free small molecular quenchers were used to bypass covalent labeling,514 and nanoparticlebased quenchers were used to achieve higher quenching efficiency and for delivery to cells.515−517 To minimize the background fluorescence from sample matrixes, magnetic separation coupled to the flow cytometric method was designed.518 Instead of measuring steady-state fluorescence intensity, the signal can be provided by fluorescence anisotropy reflecting the changes in rotational motion of the fluorophore label.519−521 Signaling does not have to rely on fluorescence. For example, two types of gold nanoparticles can be used to achieve colorimetric detection (Figure 27A). AuNPs are purple/blue in color when close to each other but red when dispersed. Cleavage of substrate triggers AuNP disassembly resulting in a blue-to-red color change.302,522,523 Magnetic nanoparticles or labels were employed to achieve distance-dependent magnetic signaling, which might be useful for in vivo MRI imaging.524,525 By labeling a methylene blue on the DNAzyme, electrochemical signaling was also demonstrated after immobilization of the DNAzyme on an electrode surface (Figure 27B).301 To achieve a surface enhanced Raman scattering (SERS) signal, the immobilized DNAzyme can be extended to further hybridize with DNA on AuNPs coated with mercaptobenzoic acid as a Raman dye (Figure 27C).526

on signaling methods. This is because signaling methods are extremely versatile for DNA due to its programmability and ease of labeling. In addition, different DNAzymes and aptamers can share the same signaling strategy. To avoid redundancy, we provide an overview of the sensor design strategies in a separate section here, but without referring to specific DNA sequences in most cases. 4.1. Catalytic Beacons

It is interesting to note that most RNA-cleaving DNAzymes have very similar secondary structures as schematically shown in Figure 26A.507 In the presence of a target metal ion, the substrate strand is cleaved into two pieces at the cleavage site. Compared to the full-length substrate, the cleaved fragments have lower affinity (and thus lower Tm) with the enzyme strand. If the Tm is below room temperature, dissociation is expected. Taking advantage of this property, Li and Lu first labeled a fluorophore on one end of the substrate strand and a fluorescence quencher on the corresponding end of the enzyme, masking the initial fluorescence (Figure 26B).27 Cleavage was then measured by the enhanced fluorescence emission. Typically, fluorescence signal is monitored as a function of time, thus the rate of signal change can be calculated. The rate reflects the intrinsic enzyme property and thus, is less dependent on the absolute fluorescence intensity. In addition, a metal ion may induce more than one cleavage reaction, so that catalytic turnovers are helpful for signal amplification at low metal concentrations. Most such catalytic beacons can detect transition metals down to low nM (low parts-per-billion) and sometimes even pM (parts-per-trillion) levels, which is difficult to achieve for pure binding based sensors. The position of labeling has been varied to improve sensor performance.508 For example, an additional quencher was added to the other end of the substrate to reduce background fluorescence from nonhybridized substrates.300 Asymmetric design was used to enhance the overall stability of the uncleaved DNAzyme complex, while reducing the stability of 8296


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Figure 28. (A) Design principle of a MB for target cDNA detection.538 (B) An aptamer beacon constructed using T-Hg2+-T or C-Ag+-C forming the stem region.542,543 (C) K+-induced the formation of a G4 structure causes an end-to-end distance change signaled by an enhanced FRET.170 (D) A scheme of structure-switching signaling aptamer design.544,545 The aptamer is extended and partially hybridized with two pieces of short labeled DNA, resulting in quenched fluorescence. In the presence of the target, the quencher-bearing DNA is released yielding fluorescence increase.

Figure 29. (A) Typical DNAzyme structure with conserved regions in green and a hairpin that can be replaced by other hairpins. (B) A Hg2+ aptazyme designed by incorporating multiple T-T mismatches to the hairpin of a UO22+-specific DNAzyme.390 (C) An aptazyme designed by inserting an aptamer sequence to the substrate binding stem of a DNAzyme.556 (D) The library design for in vitro selection of aptazymes by fixing the hammerhead ribozyme region and randomizing the aptamer region.421

4.2. Aptamer Beacons

Protein enzymes have also been used as a label, and DNAzyme cleavage can release the enzyme label to achieve highly sensitive detection due to catalytic amplification. For example, the Lu group attached an invertase. Upon cleavage, the invertase-bearing fragment is released to convert sucrose to glucose for detection by a personal glucose meter (Figure 27D).527 This method was further improved for the general detection of many heavy metal ions.528 The Li lab attached a urease to achieve a visual colorimetric detection using a pH indicating dye.529 Other detection methods based on circular dichroism,530 computer readable disks,531 and surface plasmon resonance (SPR)532 have been developed as well. DNAzymes can also be integrated into micro- and nanofluidic devices for automated and rapid testing with a very small sample volume.533−535 To improve the robustness of such sensors, such as sensitivity to temperature, two sensors with different temperature dependencies were mixed to cancel the temperature effect.536 This is not a comprehensive list of all possible signaling methods based on the catalytic beacon idea. The design principle takes advantage of the release of a cleaved fragment, and the choice of the signaling method often depends on specific applications.

Aptamer binding usually produces a conformational change,537 which is useful for sensor design. Many signaling strategies are available for aptamers as well, and the aptamer beacon idea is a popular one. The gist of an aptamer beacon is to harness DNA folding. Since the secondary structures and target binding modes are highly different among aptamers, the design strategy for aptamer beacon is also quite diverse. Early inspirations of aptamer beacons came from the commercially successful molecular beacons (MBs). A MB refers to a DNA hairpin with the two ends labeled with a fluorophore and a quencher, respectively. The quenched fluorescence is recovered by hybridizing to the target complementary DNA (cDNA, Figure 28A).538 Several groups have directly adapted this design to aptamers.539−541 For example, Tan and co-workers used T-Hg2+-T or C-Ag+-C base pairs as the stem region of a hairpin for signaling (Figure 28B).542,543 However, since metal binding needs to compete with the cDNA, its sensitivity might be limited. Another aptamer beacon is to label a FRET acceptor/donor pair on the ends; signaling then depends on the change of end-to-end distance (Figure 28C).170 It is also possible to split an aptamer into two pieces to produce an even larger end-to-end distance 8297


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Figure 30. (A) Structures of some typical fluorescent base analogs including 2-aminopurine (2AP, adenine analog), 3-methyl isoxanthopterin (3-MI, guanine analog), 6-methyl isoxanthopterin (6-MI, guanine analog) and 4-amino-6-methylpteridone (6-MAP, adenine analog).561 (B) The structure of pyrrolo-cytosine (pC, cytosine analog).561 (C) Na+ sensing based on the local folding of the Ce13d DNAzyme probed by 2AP.141 (D) Hg2+ or Ag+ sensors using 2AP labeled DNA homopolymers.567 (E) The CueR dimer binds a pC-modified promoter DNA. In the presence of Cu+, pC unwinds from base pairing with increased fluorescence.568 (F) Ag+-induced formation of the C−Ag+-pC base pair resulting in fluorescence decrease.460

change.544 However, such split aptamers sacrifice target binding affinity. A key innovation in this field is the structure-switching aptamer idea pioneered by Li and co-workers.545,546 In this design, a short piece of oligonucleotide is introduced to compete with the target molecule for aptamer binding (Figure 28D). The quencher label can fully dissociate from the fluorophore resulting in a much greater signal change compared to traditional aptamer beacons. It is also a light-up design with often greater than 10-fold signal enhancement. This method has been successfully demonstrated for detecting many targets including metal ions.92,547 The potential false signaling caused by a nonspecific probe release was elegantly solved by using a control reporter that is similarly sensitive to the buffer change but insensitive to the target analyte.548 A disadvantage of structure-switching aptamers, however, is the weakened target binding affinity due to competition from the quencher-labeled DNA. Aptamer beacons do not have to use fluorescence signaling either. Other signals, such as color,549 electrochemistry,550,551 Rayleigh scattering,552 SERS,553 resonance light scattering (RLS),554 and quantifiable digital signals555 were all demonstrated.

Since the aforementioned catalytic beacon strategy is readily adaptable to aptazymes, we only summarize the strategies for aptazyme design here. In general, a DNAzyme contains two substrate binding stems and a catalytic core (Figure 29A). Since the catalytic cores of many DNAzymes contain a hairpin that plays only a structural role, a simple way is to incorporate an aptamer into the hairpin (Figure 29B).390 Alternatively, the aptamer can be placed to modulate the stability of a substrate binding stem (Figure 29C).556 In either case, the proper folding of the DNAzyme is disrupted until the aptamer binds to its target.109,557,558 Such designs, however, are a trial-and-error process, and a few sequences may have to be tested to obtain an optimal aptazyme. In addition, the level of activation might be low. These problems can be solved by in vitro selection to achieve an optimal communication between the aptamer part and the catalytic part.559 In addition, selection of aptazymes can be a way to obtain new aptamers. For example, Breaker and coworkers isolated a series of aptazymes to target Co2+408 and other divalent ions using the hammerhead ribozyme scaffold (Figure 29D).421 Compared to conventional DNAzyme selection, aptazyme selection is based on existing enzymes and extensive negative selections might be required to ensure selectivity. Recently, a few aptazymes were selected unintentionally. For example, the Ce13d DNAzyme requires a Na+ and the catalytic role is directly performed by a lanthanide.110,134,135 The Ag10c DNAzyme binds two Ag+ ions in its aptamer pocket and the catalytic role is achieved by a group 1A or 2A metal.119,480

4.3. Aptazymes

Aptazymes refer to DNAzymes or ribozymes containing an aptamer motif, so that the catalytic activity can be controlled by aptamer binding. In a typical aptazyme, the activity is low in the absence of the target. Target binding rigidifies the aptamer structure to activate the DNAzyme. Aptazymes have the advantage of both DNAzymes and aptamers. First, since one DNAzyme can turnover multiple substrates, aptazymes allow for signal amplification. Second, since target binding is reflected by a chemical reaction, it can minimize nonspecific signal transduction, which is sometimes seen in aptamer sensors.

4.4. Signaling Based on Local Folding

Aptamer-based sensing can also be achieved by a single covalently labeled fluorophore, which is based on the premise that binding-induced folding can substantially alter the local 8298


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Figure 31. Examples of label-free DNA sensors for metal detection. (A) The cleavage of a DNAzyme in the presence of target metal dissociates an intercalating dye resulting in fluorescence decrease.571−573 (B) Metal binding releases a DNA aptamer from the CCP for fluorescence recovery.578 (C) After forming T-Hg2+-T base pairs, the DNA cannot adsorb on AuNPs, making the AuNPs more easily aggregated by salt to give a blue color.177,579,580 (D) The formation of C-Ag+-C base pairs assembles a G4 structure to catalyze the ABTS-H2O2 reaction for a colored product.583

environment of attached fluorophore and change its emission properties. The first demonstration was reported by Ellington and co-workers for ATP detection.560 However, the amount of fluorescence change was quite moderate with most designed failed to produce any signal in this trial-and-error process. This is attributable to the fact that the fluorophores used are not very sensitive to the local folding environment. An alternative way is to use fluorescent base analogs. Their fluorescence is often highly dependent on local the base stacking environment in DNA. Base analogs have relatively small perturbations on DNA structure, and some even mimic natural bases and retain the hydrogen bonding pattern. Many fluorescent base analogs have been reported for sensing applications,561−565 and some examples are shown in Figure 30A. Among them, 2-aminopurine (2AP) is a popular analog of adenine. Free 2AP has a quantum yield of 0.68 in water. When incorporated into duplex DNA, its fluorescence is strongly quenched due to stacking with neighboring bases, while after relaxing from base stacking or flipping out of the duplex, its fluorescence is significantly enhanced.566 We recently labeled a 2AP in the Ce13d DNAzyme and observed 700% fluorescence rise upon adding Na+ (Figure 30C).141 We also developed Hg2+ and Ag+ sensors by imbedding a 2AP into the middle of poly-T and poly-C DNAs achieving 14- and 10-fold fluorescence enhancements, respectively (Figure 30D).567 While 2AP is a quite robust fluorophore (e.g., insensitive to pH change), its excitation and emission are both in the UV region, limiting its biological imaging applications. This problem is common to many such base analogs. To address this, expanded analogs have been synthesized with one or more additional aromatic rings added to or inserted into the natural bases, leading to red-shifted absorption and emission spectra. One such analog is pyrrolo-cytosine (pC, a cytosine analog; Figure 30B). The pC shares the same basic fluorescent properties as 2AP, but it emits in the blue region (473 nm). Using this probe, Brown et al. reported a highly sensitive Cu+ sensor by using MerR protein as the metal recognition element, while a labeled DNA produced the signal (Figure 30E).568,569 Similar designs were also used for sensing Pb2+ and Hg2+.7,570 In another study, Park et al. discovered that pC maintained its

ability to form the pC-Ag+-C base pair (Figure 30F). Ag+ binding changes the base stacking of pC and quenches its fluorescence.460 4.5. Label-Free Methods

All of the above methods involve covalent labels. Label-free methods are attractive since they use nonmodified DNA and are therefore more cost-effective. In addition, functional DNAs can fully preserve its native activity. Many label-free methods employ a fluorescent signal. One common way is to use DNA intercalating dyes such as SYBR Green I (SGI),571 ethidium bromide (EB), 572 and picogreen (PG). 573 They bind DNAzymes with a strong initial fluorescence. After metalinduced cleavage, fluorescence is dropped (Figure 31A). Turnon sensors were designed using aptamers, since metal binding often folds aptamers into more compact structures.574,575 Some dyes that can stain abasic sites in DNA576,577 and fluorescent cationic conjugated polymers (CCP) have also been explored (Figure 31B).578 CCP emission is initially quenched by binding to an unfolded aptamer. Upon target binding, the CPP is released to become fluorescent. Aside from fluorescence, other types of signals can also be used for label-free detection. For example, AuNPs are more protected against salt-induced aggregation by unfolded aptamers, while metal binding can fold the aptamer and inhibit its protection effect. As a result, AuNPs turn a blue color upon salt addition (Figure 31C).177,579,580 Such a method was also applied to DNAzymes for metal detection based on its cleavage reaction.581,582 Another popular method is to use a G4 DNA/ hemin complex to mimic peroxidase activity catalyzing the color change of ABTS or TMB in the presence of H2O2 (Figure 31D).583 Metal binding is designed to promote formation of the required G4 structure for catalysis.584 Similarly, fluorophore binding aptamers can also be tethered for signal generation.585,586 By immobilizing an aptamer on a gold surface, metal binding can change the dielectric constant of the gold surface, leading to a surface plasmon resonance (SPR) signal.587 Binding can also cause a mass change detectable using a quartz crystal microbalance (QCM).588 8299


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Figure 32. AuNP-based lateral flow sensors for metal detection. (A) A schematic of a typical lateral flow device containing four overlapping pads. (B) Hg2+-induced disassembly of AuNPs, and the released AuNPs are captured via biotin−streptavidin interactions at the testing zone on the membrane.590 (C) Pb2+-induced substrate cleavage by a DNAzyme, and the cleaved fragment on the AuNP surface was captured by the immobilized cDNA on the membrane.591

site monitoring. Sensors can potentially achieve this for their excellent sensitivity and selectivity. Since their general analytical applications are quite obvious, we herein highlight a few emerging applications that have attracted recent attention.

However, label-free methods may suffer from false positive results. For example, for those methods based on the mass change or refractive index change, nonspecific binding is always a practical problem especially in complex sample matrix such as serum.589 In addition, in real samples, noncovalent labels (e.g., fluorophore, AuNPs) might be sequestered by proteins or other molecules.

5.1. Metal Speciation

Metal speciation information is critical for understanding environmental chemistry and biology. For example, Cr3+ is less toxic compared to Cr(VI). Hg2+ and organomercury also have different toxicities. Metal ions associated with dissolved organic matters (DOM) are less bioavailable and thus can be detoxified.593 Other speciation information important in biology includes Fe2+/Fe3+ and Cu2+/Cu+. Since Fe2+ and Cu+ are unstable at ambient conditions, they are only important in biological systems. However, most instrumentation methods only measure the total metal concentration without specifying the oxidation state. In this regard, biosensors might be more useful since they can provide such information. For example, the Ce13d DNAzyme works only with Ce3+ but not Ce4+.110 Therefore, speciation information can be obtained by first measuring Ce3+ in the sample. Then Ce4+ is reduced to Ce3+ using iodide, and a second measurement can be performed to obtain the total cerium concentration. The difference of the two measurements is the Ce4+. Similarly, Cr(VI) was reduced to Cr3+ using NaBH4 to be detected by a DNAzyme.447 In these examples, speciation analysis relies on a redox reaction. While this in principle should work, a few problems might arise. (1) The reducing agent might affect sensor performance. (2) A trace amount of analyte might not be effectively reduced or detected by the difference of the two measurements. (3) A larger error might be introduced as a subtraction step is required. In an ideal case, two sensors are needed, each responding only to a particular oxidation state. This allows the measurement of both species without additional oxidation/reduction. Such an example has yet to be demonstrated. The closest system might be DNAzyme-based copper detection. A DNAcleaving DNAzyme detects copper in the presence of ascorbate, where Cu2+ is likely converted to Cu+ for activity.351

4.6. Lateral Flow Devices

Lateral flow technology is commonly used for antibody-based detection. It is appealing because of its simplicity. To develop lateral flow based sensors using aptamers, AuNPs were often used for color display due to its ultrahigh extinction coefficient. In a typical lateral flow device, four overlapping pads are placed on a backing, and they are the (from bottom to top) wicking pad, glass fiber conjugation pad, membrane ,and absorption pad (Figure 32A). The wicking pad is designed for sample loading; sensors are spotted on the conjugation pad; and the signal is observed on the membrane. Using lateral flow technology for metal detection has not been extensively explored. In one study, Torabi and Lu attempted Hg2+ detection by assembling two types of DNAfunctionalized AuNPs using a linker with a T-rich region, so that the assembly could be dissociated by adding Hg2+ due to thymine-Hg2+ binding. One of the DNA on AuNPs was labeled with a biotin, which can be captured at the test zone through the biotin−streptavidin interaction (Figure 32B).590 A DNAzyme was also used by the Lu group. They elongated the 5′-end of the substrate of the 8-17 DNAzyme to attach on AuNPs. The same DNA was also used for hybridization with the capture DNA at the testing zone (Figure 32C).591 In the presence of Pb2+, the substrate was cleaved, releasing the AuNPs to be captured. Similar work was also carried out by Fang et al., who developed a DNAzyme-based Cu2+ sensor.592

5. EMERGING APPLICATIONS Measuring metal ions is urgently needed for various applications. The goal of such DNA-based metal sensors is to partially replace traditional analytical instruments for initial on 8300


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Interestingly, an RNA-cleaving DNAzyme is inhibited by adding ascorbate; thus, it likely only uses Cu2+.107 Although these two DNAzymes use copper of different oxidation states, a reducing agent, ascorbate, is still involved. Most of such speciation studies are in the proof-of-concept stage without being applied to practical samples. We believe this is an important research front that could complement instrumental analysis methods. So far, we only considered speciation due to redox reactions. At the same time, DNA-based sensors do not have a very high binding affinity (e.g., Kd = 1 μM or higher). In real samples, DNA must compete with other ligands for the metal. For example, in environmental samples, DOM such as humic acid may compete with DNA. Even some inorganic ligands such as chloride can coordinate with metal ions such as Hg2+ to decrease its available concentration to DNA. Metal hydrolysis is another dimension of metal speciation, which further decreases the concentration of free metals. For biological samples, this problem is even more serious since concentrated proteins and other molecules can strongly bind various metals. Therefore, measurement results from DNA-based metal sensors need to be carefully interpreted.

Table 3. Regulated Metal Contaminants in Drinking Water Set by the U.S. EPA, and Their Detection by DNA-Based Sensorsa metal

maximum contaminant level (nM)

antimony barium

50 14560

beryllium cadmium

440 40

chromium (total)

1920

copper

20,460

5.2. Environmental Monitoring

Monitoring metals in the environment is a long-standing analytical task. Heavy metal outbreaks are often seen in the news. Therefore, both government agencies and common household users need to have reliable and cost-effective metal sensors. Table 3 shows the maximal contamination concentrations of some metal ions in drinking water set by the US EPA, and most of these concentrations can be detected by DNA-based sensors, supporting the potential environmental impact of such sensors.594,595 Technically, without concentrated proteins in the matrix, environmental water samples are less complicated compared to biological samples. Indeed, most of the work presented in this review involves river, lake, and even ocean samples.596 Still, the effect of DOM needs to be carefully studied since they can also chelate metal ions. Other environmental samples include soil, dust, and paint, just to name a few. For these solid samples, a standard method is followed to extract metals into aqueous solutions for analysis. For real sample detection, the ionic strength and temperature may affect the performance of DNAbased sensors. To meet the demand of different users, sensors should be robust enough to work in a diverse range of environmental conditions. While most research in academic laboratories are only for proof-of-concept purposes, there are also examples of commercialized DNAzyme-based sensors. ANDalyze Inc. based in Champaign, IL (USA) developed a portable fluorometer to read the kinetics of DNAzyme-based sensor signals, thus bypassing the calibration problem of fluorescence intensity based measurements. Sensors for a few common heavy metals have obtained the EPA certification and are commercially available now. As explained in the previous section regarding metal speciation, we expect a difference between the concentrations measured by analytical instruments such as ICP-MS and by the sensors. ICP measures the total metal concentration, while the sensors measure only the soluble free metals at a particular oxidation state. Therefore, it is unlikely that such sensors can fully replace ICP, but rather, they can serve as an initial screen.

lead

70

mercury (inorganic)

10

thallium

10

sensing DNA NA G4 DNA ODFs ODFs Cd16 DNAzyme; Cd4 aptamer Cd-2−2 aptamer ODFs Ce13d DNAzyme ODFs DNAcleaving DNAzyme, PSCu10 DNAzyme; Cu100 DNA GpG-duplex DNA ODFs GR5 DNAzyme; 8-17 DNAzyme G4 DNA ODFs T-rich DNA PS DNA ODFs G-rich DNA (Tl+) Tm7 DNAzyme (PS substrate) ODFs

detection limit (nM)

ref

0.8 105 5000 1.1

168 273 273 106

4.6 40

94 93

100 Cr(III):70 Cr(VI):140 Cr(III)/Cr(VI):100 35

597 447 597 351

1.6

107

87 1.2

358 355

5000 3.7

273 97

7.8

97

1 5000 40 1.7 100 4600 (Tl+)

321 273 65 399 597 334

1.5 (Tl+)

336

1000 (Tl+)

273

a

ODFs denote oligodeoxyfluoroside chemosensors (see section 6.2 for details), and NA indicates not available.

ICP can be used for confirmation and for more accurate measurements. 5.3. Caged Aptamers/DNAzymes

Aside from environmental applications, it is also desirable to measure intracellular metal concentrations with a high spatial and temporal resolution. Within this context, caged DNAzymes are quite useful. In a caged molecule, a photolabile group is covalently attached to block its activity. Upon shining a light of a particular wavelength, the capping group falls off and the molecule becomes active again.598 This idea is quite useful since light can be applied conveniently both in cell cultures and in tissues with precisely controlled amplitude, location, and timing. Caged aptamers were designed by capping crucial nucleobases responsible for target binding (Figure 33A, D).599 This method is also applicable to DNAzymes by modifying the conserved nucleobases at the catalytic loop to inhibit the DNAzyme activity (Figure 33A,E).600 Caging can also be made 8301


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Figure 33. Caged aptamers/DNAzymes. The caging groups can be attached to (A) nucleobases,599−602 (B) the 2′-OH group,105,603,604 and (C) the phosphate backbone.225,605 The photolabile bonds were highlighted in red. Caging groups are incorporated to the essential nucleotides to inactive (D) an aptamer599 and (E) a DNAzyme.600 (F) Caging groups are modified at the substrate binding arm to unwind the base pairing.601 (G) A cage group is introduced to the 2′-OH group at the cleavage site to deactivate the DNAzyme.604

Table 4. Concentrations of Several Most Abundant Metals in Biological Fluids cation

serum (mM)

intracellular matrix (mM)

urine (mM)

ref.

Na+ K+ Ca2+ Mg2+ Zn2+ Cu2+ Fe2+/Fe3+ (total)

142 4.3 2.5 1.1 (free) 0.011 0.015 0.026 ± 0.013

12 139 100 mM), Na+ (∼10 mM), and Mg2+ (∼1 mM free Mg2+), while Ca2+ exists only at a trace level (

Metal Sensing by DNA - ACS Publications - American Chemical Society

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