Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme–Catalytic Hairpin Assembly Probe

DNAzymes are a promising platform for metal ion detection, and a few DNAzyme‐based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na⁺‐specific DNAzyme to detect endogenous Na⁺ inside cells is reported. Upon light activation and in the presence of Na⁺, the NaA43 DNAzyme cleaves its substrate strand and releases a product strand, which becomes an initiator that trigger the subsequent CHA amplification reaction. This strategy allows detection of endogenous Na⁺ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems.     

Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme–Catalytic Hairpin Assembly Probe

DNAzymes are a promising platform for metal ion detection, and a few DNAzyme-based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na⁺-specific DNAzyme to detect endogenous Na⁺ inside cells is reported. Upon light activation and in the presence of Na⁺, the NaA43 DNAzyme cleaves its substrate strand and releases a product strand, which becomes an initiator that trigger the subsequent CHA amplification reaction. This strategy allows detection of endogenous Na⁺ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems.     

Near‐Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal‐dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near‐infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three‐stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal‐ion‐dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Sharp Switching of DNAzyme Activity through the Formation of a CuII-Mediated Carboxyimidazole Base Pair

DNAzymes are widely used as functional units for creating DNA-based sensors and devices. Switching of DNAzyme activity by external stimuli is of increasing interest. Herein we report a Cu^II-responsive DNAzyme rationally designed by incorporating one of the most stabilizing artificial metallo-base pairs, a Cu^II-mediated carboxyimidazole base pair (Im^C-Cu^II-Im^C), into a known RNA-cleaving DNAzyme. Cleavage of the substrate was suppressed without Cu^II, but the reaction proceeded efficiently in the presence of Cu^II ions. This is due to the induction of a catalytically active structure by Im^C-Cu^II-Im^C pairing. The on/off ratio was as high as 12-fold, which far exceeds that of the previously reported DNAzyme with a Cu^II-mediated hydroxypyridone base pair. The DNAzyme activity can be regulated specifically in response to Cu^II ions during the reaction through the addition, removal, or reduction of Cu^II. This approach should advance the development of stimuli-responsive DNA systems with a well-defined sharp switching function.     

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×10¹⁰ bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg²⁺ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg²⁺. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg²⁺ in HeLa cells.     

Near-Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal-dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near-infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three-stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal-ion-dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Intelligent demethylase-driven DNAzyme sensor for highly reliable metal-ion imaging in living cells

The accurate intracellular imaging of metal ions requires an exquisite site-specific activation of metal-ion sensors, for which the pervasive epigenetic regulation strategy can serve as an ideal alternative thanks to its orthogonal control feature and endogenous cell/tissue-specific expression pattern. Herein, a simple yet versatile demethylation strategy was proposed for on-site repairing-to-activating the metal-ion-targeting DNAzyme and for achieving the accurate site-specific imaging of metal ions in live cells. This endogenous epigenetic demethylation-regulating DNAzyme system was prepared by modifying the DNAzyme with an m⁶A methylation group that incapacitates the DNAzyme probe, thus eliminating possible off-site signal leakage, while the cell-specific demethylase-mediated removal of methylation modification could efficiently restore the initial catalytic DNAzyme for sensing metal ions, thus allowing a high-contrast bioimaging in live cells. This epigenetic repair-to-activate DNAzyme strategy may facilitate the robust visualization of disease-specific biomarkers for in-depth exploration of their biological functions.

A simple yet versatile demethylation strategy is proposed for an on-site repairing-to-activating metal-ion-targeting DNAzyme and for achieving the highly reliable site-specific imaging of metal ions in live cells.     

Modulation of DNAzyme Activity via Butanol Dehydration

Understanding the activity of biomolecules in cosolvent systems is important for catalysis, separation and developing biosensors. The majority of previously studied solvents are either phase separated with water or miscible with water. Butanol was recently used to extract water for the conjugation of DNA to gold nanoparticles. In this work, the effect of butanol on the activity of a few RNA-cleaving DNAzymes was studied. A 130-fold improvement in sensitivity for the Na⁺-specific EtNa DNAzyme was observed, and butanol also improved the activity of another Na⁺-specific DNAzyme, NaA43T by a few folds. However, when divalent metal ions were used for both EtNa and 17E DNAzymes, the activity was inhibited. A main driven force for enhanced DNAzyme activity is the concentration effect due to butanol dehydration. This study provides insights into the interplay between DNA, metal ions and organic solvents, and such an understanding might be useful for developing sensitive biosensors.     

An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function

Engineering functional nucleic acids that are active under unusual conditions will not only reveal their hidden abilities but also lay the groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were “compelled” to accept 35 % dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35 % DMSO. This experiment led to the discovery of a new DNAzyme that requires 35 % DMSO for its catalytic activity and exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis, and its activity is enhanced by monovalent ions. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional, organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of catalytic DNA.     

Design of Ratiometric Fluorescent Probes Based on Arene–Metal‐Ion Interactions and Their Application to CdII and Hydrogen Sulfide Imaging in Living Cells

Non‐coordinative interactions between a metal ion and the aromatic ring of a fluorophore can act as a versatile sensing mechanism for the detection of metal ions with a large emission change of fluorophores. We report the design of fluorescent probes based on arene–metal‐ion interactions and their biological applications. This study found that various probes having different fluorophores and metal binding units displayed significant emission redshift upon complexation with metal ions, such as Ag^I, Cd^II, Hg^II, and Pb^II. X‐ray crystallography of the complexes confirmed that the metal ions were held in close proximity to the fluorophore to form an arene–metal‐ion interaction. Electronic structure calculations based on TDDFT offered a theoretical basis for the sensing mechanism, thus showing that metal ions electrostatically modulate the energy levels of the molecular orbitals of the fluorophore. A fluorescent probe was successfully applied to the ratiometric detection of the uptake of Cd^II ions and hydrogen sulfide (H₂S) in living cells. These results highlight the utility of interactions between arene groups and metal ions in biological analyses.     

Photoactivatable Engineering of CRISPR/Cas9-Inducible DNAzyme Probe for In Situ Imaging of Nuclear Zinc Ions

DNAzyme-based fluorescent probes for imaging metal ions in living cells have received much attention recently. However, employing in situ metal ions imaging within subcellular organelles, such as nucleus, remains a significant challenge. We developed a three-stranded DNAzyme probe (TSDP) that contained a 20-base-pair (20-bp) recognition site of a CRISPR/Cas9, which blocks the DNAzyme activity. When Cas9, with its specialized nuclear localization function, forms an active complex with sgRNA within the cell nucleus, it cleaves the TSDP at the recognition site, resulting in the in situ formation of catalytic DNAzyme structure. With this design, the CRISPR/Cas9-inducible imaging of nuclear Zn²⁺ is demonstrated in living cells. Moreover, the superiority of CRISPR-DNAzyme for spatiotemporal control imaging was demonstrated by integrating it with photoactivation strategy and Boolean logic gate for dynamic monitoring nuclear Zn²⁺ in both HeLa cells and mice. Collectively, this conceptual design expands the DNAzyme toolbox for visualizing nuclear metal ions and thus provides new analytical methods for nuclear metal-associated biology.     

Metal-ion-dependent folding of a uranyl-specific DNAzyme: insight into function from fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO₂²⁺)‐specific 39E DNAzyme in the presence of Mg²⁺, Zn²⁺, Pb²⁺, or UO₂²⁺. At pH 5.5 and physiological ionic strength (100 mM Na⁺), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg²⁺ or Zn²⁺. However, no folding occurred in the presence of Pb²⁺ or UO₂²⁺; this is analogous to the “lock‐and‐key” catalysis mode first observed in the Pb²⁺‐specific 8–17 DNAzyme. However, Mg²⁺ and Zn²⁺ exert different effects on the 8–17 and 39E DNAzymes. Whereas Mg²⁺ or Zn²⁺‐dependent folding promoted 8–17 DNAzyme activity, the 39E DNAzyme folding induced by Mg²⁺ or Zn²⁺ inhibited UO₂²⁺‐specific activity. Group IIA series of metal ions (Mg²⁺, Ca²⁺, Sr²⁺) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr²⁺ (1.12 Å) is comparable to that of Pb²⁺ (1.20 Å), but contrary to Pb²⁺, Sr²⁺ induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb²⁺ and UO₂²⁺. Under low ionic strength (30 mM Na⁺), all four metal ions (Mg²⁺, Zn²⁺, Pb²⁺, and UO₂²⁺), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA‐backbone phosphates in addition to playing specific catalytic roles. Mg²⁺ at low (<2 mM ) concentration promoted UO₂²⁺‐specific activity, whereas Mg²⁺ at high (>2 mM ) concentration inhibited the UO₂²⁺‐specific activity. Therefore, the lock‐and‐key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock‐and‐key mode only at ionic strengths of 100 mM or greater.     

Orthogonal Activation of RNA‐Cleaving DNAzymes in Live Cells by Reactive Oxygen Species

RNA‐cleaving DNAzymes are useful tools for intracellular metal‐ion sensing and gene regulation. Incorporating stimuli‐responsive modifications into these DNAzymes enables their activities to be spatiotemporally and chemically controlled for more precise applications. Despite the successful development of many caged DNAzymes for light‐induced activation, DNAzymes that can be intracellularly activated by chemical inputs of biological importance, such as reactive oxygen species (ROS), are still scarce. ROS like hydrogen peroxide (H₂O₂) and hypochlorite (HClO) are critical mediators of oxidative stress‐related cell signaling and dysregulation including activation of immune system as well as progression of diseases and aging. Herein, we report ROS‐activable DNAzymes by introducing phenylboronate and phosphorothioate modifications to the Zn²⁺‐dependent 8–17 DNAzyme. These ROS‐activable DNAzymes were orthogonally activated by H₂O₂ and HClO inside live human and mouse cells.     

A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing

DNAzymes hold promise for gene‐silencing therapy, but the lack of sufficient cofactors in the cell cytoplasm, poor membrane permeability, and poor biostability have limited the use of DNAzymes in therapeutics. We report a DNAzyme–MnO₂ nanosystem for gene‐silencing therapy. MnO₂ nanosheets adsorb chlorin e6‐labelled DNAzymes (Ce6), protect them from enzymatic digestion, and efficiently deliver them into cells. The nanosystem can also inhibit ¹O₂ generation by Ce6 in the circulatory system. In the presence of intracellular glutathione (GSH), MnO₂ is reduced to Mn²⁺ ions, which serve as cofactors of 10–23 DNAzyme for gene silencing. The release of Ce6 generates ¹O₂ for more efficient photodynamic therapy. The Mn²⁺ ions also enhance magnetic resonance contrast, providing GSH‐activated magnetic resonance imaging (MRI) of tumor cells. The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.     

In vitro Selection of Chemically Modified DNAzymes

DNAzymes are in vitro selected DNA oligonucleotides with catalytic activities. RNA cleavage is one of the most extensively studied DNAzyme reactions. To expand the chemical functionality of DNA, various chemical modifications have been made during and after selection. In this review, we summarize examples of RNA-cleaving DNAzymes and focus on those modifications introduced during in vitro selection. By incorporating various modified nucleotides via polymerase chain reaction (PCR) or primer extension, a few DNAzymes were obtained that can be specifically activated by metal ions such as Zn²⁺ and Hg²⁺. In addition, some modifications were introduced to mimic RNase A that can cleave RNA substrates in the absence of divalent metal ions. In addition, single modifications at the fixed regions of DNA libraries, especially at the cleavage junctions, have been tested, and examples of DNAzymes with phosphorothioate and histidine-glycine modified tertiary amine were successfully obtained specific for Cu²⁺, Cd²⁺, Zn²⁺, and Ni²⁺. Labeling fluorophore/quencher pair right next to the cleavage junction was also used to obtain signaling DNAzymes for detecting various metal ions and cells. Furthermore, we reviewed work on the cleavage of 2′-5′ linked RNA and L-RNA substrates. Finally, applications of these modified DNAzymes as biosensors, RNases, and biochemical probes are briefly described with a few future research opportunities outlined at the end.     

Graphene–DNAzyme junctions: a platform for direct metal ion detection with ultrahigh sensitivity

Many metal ions are present in biology and in the human body in trace amounts. Despite numerous efforts, metal sensors with ultrahigh sensitivity (<a few picomolar) are rarely achieved. Here, we describe a platform method that integrates a Cu²⁺-dependent DNAzyme into graphene–molecule junctions and its application for direct detection of paramagnetic Cu²⁺ with femtomolar sensitivity and high selectivity. Since DNAzymes specific for other metal ions can be obtained through in vitro selection, the method demonstrated here can be applied to the detection of a broad range of other metal ions.     

Caging-Decaging Strategies to Realize Spatiotemporal Control of DNAzyme Activity for Biosensing and Bioimaging

DNAzymes with RNA-cleaving activity have been widely used as biosensing and bioimaging tools for detection of metal ions. Despite the achievements, DNAzyme-based biosensors sometime suffer from false positive signals and unexpected off-target turn-on in biological environments, which are likely due to the unstable nature of the RNA site. Ways to control DNAzyme activity in order to improve the sensing performance remain a significant challenge. To meet the challenge, there is growing interest to develop synthetic strategies that can cage native DNAzyme under undesired conditions and reactivate it in target environment in order to function in a controlled manner. A variety of caging-decaging strategies have been developed to realize spatiotemporal control of the DNAzyme activity, improving its specificity and sensitivity as well as extending its application regimes. In this review, we focus on strategies to regulate the catalytic activity of DNAzyme, highlight the nucleic acid modification chemistries, and summarize three strategies to cage DNAzyme functions. Examples of using caged DNAzyme for bio-applications have also been reviewed in detail. Finally, we provide our perspectives on the potential challenges and opportunities of this emerging research topic that could advance the DNAzyme field.     

Boronic Acid-Mediated Activity Control of Split 10–23 DNAzymes

The 10–23 DNAzyme is an artificially developed Mg²⁺-dependent catalytic oligonucleotide that can cleave an RNA substrate in a sequence-specific fashion. In this study, new split 10–23 DNAzymes made of two nonfunctional fragments, one of which carries a boronic acid group at its 5′ end, while the other has a ribonucleotide at its 3′ end, were designed. Herein it is demonstrated that the addition of Mg²⁺ ions leads to assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation identified the best-performing system. The results highlight key features for efficient control of DNAzyme activity through the formation of boronate linkages.     

DNAzyme‐Loaded Metal–Organic Frameworks (MOFs) for Self‐Sufficient Gene Therapy

DNAzymes have been recognized as potent therapeutic agents for gene therapy, while their inefficient intracellular delivery and insufficient cofactor supply precludes their practical biological applications. Metal–organic frameworks (MOFs) have emerged as promising drug carriers without in‐depth consideration of their disassembled ingredients. Herein, we report a self‐sufficient MOF‐based chlorin e6‐modified DNAzyme (Ce6‐DNAzyme) therapeutic nanosystem for combined gene therapy and photodynamic therapy (PDT). The ZIF‐8 nanoparticles (NPs) could efficiently deliver the therapeutic DNAzyme without degradation into cancer cells. The pH‐responsive ZIF‐8 NPs disassemble with the concomitant release of the guest DNAzyme payloads and the host Zn²⁺ ions that serve, respectively, as messenger RNA‐targeting agent and required DNAzyme cofactors for activating gene therapy. The auxiliary photosensitizer Ce6 could produce reactive oxygen species (ROS) and provide a fluorescence signal for the imaging‐guided gene therapy/PDT.     

Stabilizing DNAzymes through Encapsulation in a Metal–Organic Framework

DNAzymes are a promising class of bioinspired catalyst; however, their structural instability limits their potential. Herein, a method to stabilize DNAzymes by encapsulating them in a metal–organic framework (MOF) host is reported. This biomimetic mineralization process makes DNAzymes active under a wider range of conditions. The concept is demonstrated by encapsulating hemin-G-quadruplex (Hemin-G4) into zeolitic imidazolate framework-90 (ZIF-90), which indeed increases the DNAzyme's structural stability. The stabilized DNAzymes show activities in the presence of Exonuclease I, organic solvents, or high temperature. Owing to its elevated stability and heterogeneous nature, it is possible to perform catalysis under continuous-flow conditions, and the DNAzyme can be reactivated in situ by introducing K⁺. Moreover, it is found that the encapsulated DNAzyme maintains its high enantiomer selectivity, demonstrated by the sulfoxidation of thioanisole to (S)-methyl phenyl sulfoxide. This concept of stabilizing DNAzymes expands their potential application in chemical industry.