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.     

Ultrasensitive detection of lead ion based on target induced assembly of DNAzyme modified gold nanoparticle and graphene oxide

In this paper, we report a novel colorimetric strategy for the detection of small molecules by using Pb²⁺ ion as an example. In this strategy, DNAzyme duplex modified gold nanoparticles (GNPs) are designed to be unable to interact with graphene oxide (GO). However, in the presence of Pb²⁺, the substrate strand of the DNAzyme is cleaved at its cleavage site, resulting in the disassembly of the DNAzyme duplex modified GNPs into three parts, i.e., the 3′- and 5′-fragments of substrate strand and the DNAzyme strand modified GNPs. By taking advantage of the efficient cross-linking effect of ssDNA-GNPs to GO, colorimetric sensor for the detection of the metal ion can be fabricated with a detection limit of 100 pM, which is much lower than the previous reports. This colorimetric method has also been used for the determination of Pb²⁺ in the tap water of the local city and the water from a reservoir with satisfactory results, so it may have potential applications in the future.     

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.     

Nucleic Acid Driven DNA Machineries Synthesizing Mg2+‐Dependent DNAzymes: An Interplay between DNA Sensing and Logic‐Gate Operations

Polymerase/nicking enzymes and nucleic‐acid scaffolds are implemented as DNA machines for the development of amplified DNA‐detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase‐induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg²⁺‐dependent DNAzyme or the Mg²⁺‐dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher‐functionalized nucleic‐acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled‐AND gates, and the DNA‐analyte targets represent two nucleic acid sequences of the smallpox viral genome.     

Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg²⁺-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg²⁺ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10⁻⁴ U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.     

Engineering a DNA-cleaving DNAzyme and PCR into a simple sensor for zinc ion detection

The development of a simple sensor (9NL27-Zn) based on DNAzyme and PCR and aimed at the detection of low concentrations of zinc (II) ions is described. A specific Zn(II)-dependent DNAzyme (9NL27) with DNA-cleaving activity was employed. In the presence of zinc (II), the DNAzyme hydrolyzed DNA substrate into two pieces (5′ and 3′ fragments), forming 3′-terminal hydroxyl in the 5′ fragment and 5′-phosphate in the 3′ fragments. Subsequently, the 5′ fragment left the DNAzyme and bound a short DNA template. The 5′ fragment was used as a primer and extended a single-stranded full-length template by Taq polymerase. Finally, this full-length template was amplified by PCR. The amplified products had a quantitative relationship with Zn(II) concentration. Under our experimental conditions, the DNA sensor showed sensitivity (10 nM) and high specificity for zinc ion detection. After improvement of the DNA sensor, the detection limit can reach 1 nM. The simple DNA sensor may become a DNA model for the detection of trace amounts of other targets.

Detection of Metal Ions (Cu2+, Hg2+) and Cocaine by Using Ligation DNAzyme Machinery

The Cu²⁺‐dependent ligation DNAzyme is implemented as a biocatalyst for the colorimetric or chemiluminescence detection of Cu²⁺ ions, Hg²⁺ ions, or cocaine. These sensing platforms are based on the structural tailoring of the sequence of the Cu²⁺‐dependent ligation DNAzyme for specific analytes. The tethering of a subunit of the hemin/G‐quadruplex DNAzyme to the ligation DNAzyme sequence, and the incorporation of an imidazole‐functionalized nucleic‐acid sequence, which acts as a co‐substrate for the ligation DNAzyme that is tethered to the complementary hemin/G‐quadruplex subunit. In the presence of different analytes, Cu²⁺ ions, Hg²⁺ ions, or cocaine, the pretailored Cu²⁺‐dependent ligation DNAzyme sequence stimulates the respective ligation process by combining the imidazole‐functionalized co‐substrate with the ligation DNAzyme sequence. These reactions lead to the self‐assembly of stable hemin/G‐quadruplex DNAzyme nanostructures that enable the colorimetric analysis of the substrate through the DNAzyme‐catalyzed oxidation of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid), ABTS^2?, by H₂O₂ into the colored product ABTS^.?, or the chemiluminescence detection of the substrate through the DNAzyme‐catalyzed oxidation of luminol by H₂O₂. The detection limits for the sensing of Cu²⁺ ions, Hg²⁺ ions, and cocaine correspond to 1 nM , 10 nM and 2.5 μM , respectively. These different sensing platforms also reveal impressive selectivities.     

G-quadruplex-assisted enzyme strand recycling for amplified label-free fluorescent detection of UO22+

A G-quadruplex-assisted enzyme strand recycling strategy was developed for amplified label-free fluorescent detection of uranyl ion (UO₂²⁺).     

Programmed Synthesis by Stimuli‐Responsive DNAzyme‐Modified Mesoporous SiO2 Nanoparticles

DNAzyme‐capped mesoporous SiO₂ nanoparticles (MP SiO₂ NPs) are applied as stimuli‐responsive containers for programmed synthesis. Three types of MP SiO₂ NPs are prepared by loading the NPs with Cy3‐DBCO (DBCO=dibenzocyclooctyl), Cy5‐N₃, and Cy7‐N₃, and capping the NP containers with the Mg²⁺, Zn²⁺, and histidine‐dependent DNAzyme sequences, respectively. In the presence of Mg²⁺ and Zn²⁺ ions as triggers, the respective DNAzyme‐capped NPs are unlocked, leading to the “click” reaction product Cy3‐Cy5. In turn, in the presence of Mg²⁺ ions and histidine as triggers the second set of DNAzyme‐capped NPs is unlocked leading to the Cy3‐Cy7 conjugated product. The unloading of the respective NPs and the time‐dependent formation of the products are followed by fluorescence spectroscopy (FRET). A detailed kinetic model for the formation of the different products is formulated and it correlates nicely with the experimental results.     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

Target-responsive DNAzyme hydrogel for portable colorimetric detection of lanthanide(III) ions

Lanthanide elements (Ln) play an important role in industry and agriculture. As a result of the increasing consumption of lanthanides, environmental emission of Ln has become detrimental to the health of flora and fauna. Current methods for trace lanthanides detection mainly rely on sophisticated instruments. In this article, a Ln³⁺ dependent DNAzyme was incorporated into a hydrogel to generate Ln³⁺ sensitive DNAzyme hydrogel for portable colorimetric detection. The enzyme strand and its substrate strand act as crosslinker and functional unit of the hydrogel with polyacrylamide chains as the scaffold and gold nanoparticles (AuNPs) as the indicator of hydrogel stability. Any ions in the Ln³⁺ series can trigger the cleavage of substrate strand by activating the enzyme strand, thereby decreasing the crosslink ratio and leading to collapse of the hydrogel. The release of the encapsulated AuNPs turns the supernatant wine red. Using this colorimetric method, Ln³⁺ can be detected with high sensitivity, with a limit of detection (LOD) of 20 nM for Ce³⁺. The hydrogel responds specifically to any Ln³⁺ ion and works well with the spiked lake sample without the need of instruments and skilled operators. Our results suggest that the lanthanide responsive hydrogel can be used for portable and sensitive detection of Ln³⁺ contamination in the field.     

N-Methyl-4-hydrazino-7-nitrobenzofurazan: a fluorogenic substrate for peroxidase-like DNAzyme,and its potential application

Characterization and optimization studies of N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) as a new fluorogenic substrate in the peroxidation reaction catalyzed by DNAzyme are reported. The effects of pH, H₂O₂ concentration, metal-cation type, and the concentration and type of surfactant on the fluorescence intensity were investigated. The optimized reaction was subsequently used for the development of an assay for DNA detection based on a molecular-beacon probe. The use of a fluorogenic substrate enabled the detection of a single-stranded DNA target with a 1 nmol L^?1 detection limit. Graphical Abstract

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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.     

Ultrasensitive immunoassay of microcystins-LR using G-quadruplex DNAzyme as an electrocatalyst

An electrochemical immunoassay for microcystin-LR (MC-LR) detection was developed using multi-labeled horseradish peroxidase-mimicking DNAzyme on carbon nanotubes (CNTs) as electrocatalyst for signal amplification. CNTs were covalently conjugated to multiple DNAzyme along with MC-LR for a competitive immunoassay. The as-prepared DNAzyme/CNTs/MC-LR biolabel was specifically captured on the electrode surface, and current responses were obtained upon the electro-catalytic reduction of hydrogen peroxide by the captured biolabels. Under optimal conditions, the electro-catalytic current decreased linearly with the increase amount of MC-LR in the range from 0.01 to 7.0 µg L^?1. The linear regression equation was I (µA) = 12.96 ? 1.48 X _[MC–LR] (µg L^?1), with a correlation coefficient of 0.989. The limit of detection of MC-LR was 2.31 ng L^?1. Application of the immunoassay method and LC/MS/MS method for MC-LR determination on spiked reservoir water gave recovery range of 91.7–105.2% and 94.0–105.0%, respectively. The resulting versatile immunoassay exhibited high sensitivity, good precision and satisfactory reproducibility, which could have vast potential in routine water quality monitoring for various environmental toxins.     

A new Schiff base fluorescent indicator for the detection of Mg<Superscript>2+</Superscript> and its application to real samples

A new Schiff base fluorescence probe, 3-Allylsalicylaldehyde salicylhydrazone (L), for Mg²⁺ was designed and synthesized. The fluorescence of the sensor L was enhanced remarkably by Mg²⁺ with 2:1 binding ratio, and the binding constant was determined to be 1.02 × 10⁷ M^?1. Probe L had high sensitivity for Mg²⁺ in a solution of DMF/water (4:1, v/v, pH 7.5), and the detection limit was 4.88 × 10^?8 mol/L. Common coexistent metal ions, such as K⁺, Na⁺, Ag⁺, Ca²⁺, Zn²⁺, Ba²⁺, Bi²⁺, Cu²⁺, Ni²⁺, Hg²⁺, Fe³⁺ , and Al³⁺, showed little or no interference on the detection of Mg²⁺ in solution. The fluorescence probe L, which was successfully used for the determination of trace Mg(II) in real samples, was shown to be promising for liquid-phase extraction coupled with fluorescence spectra.     

Single‐Molecule Visualization of the Activity of a Zn2+‐Dependent DNAzyme

We demonstrate the single‐molecule imaging of the catalytic reaction of a Zn²⁺‐dependent DNAzyme in a DNA origami nanostructure. The single‐molecule catalytic activity of the DNAzyme was examined in the designed nanostructure, a DNA frame. The DNAzyme and a substrate strand attached to two supported dsDNA molecules were assembled in the DNA frame in two different configurations. The reaction was monitored by observing the configurational changes of the incorporated DNA strands in the DNA frame. This configurational changes were clearly observed in accordance with the progress of the reaction. The separation processes of the dsDNA molecules, as induced by the cleavage by the DNAzyme, were directly visualized by high‐speed atomic force microscopy (AFM). This nanostructure‐based AFM imaging technique is suitable for the monitoring of various chemical and biochemical catalytic reactions at the single‐molecule level.     

Assistant deoxyribonucleic acid recycling with Zn and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn²⁺ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn²⁺. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn²⁺-based amplification. The Zn²⁺ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.     

Direct detection of OTA by impedimetric aptasensor based on modified polypyrrole-dendrimers

Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 μg L⁻¹ of OTA and a detection limit of 2 ng L⁻¹ of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association.     

Combing DNAzyme with single-walled carbon nanotubes for detection of Pb(II) in water

A sensitive and simple assay for the detection of Pb(2+) in aqueous solutions is reported. It takes advantage of the high affinity between single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWCNT) as well as the capability of SWCNT in fluorescence quenching. Lead(II) catalyzes the cleavage of a fluorescently labeled DNA substrate by a DNAzyme, which releases the single-stranded product to be adsorbed onto a SWCNT. The decrease in fluorescence is proportional to the Pb(2+) concentration. Concentrations as low as 1 nM Pb(2+) in water could be detected and the detection range spans over 5 orders of magnitude. The unique combination of Pb-specific DNAzyme with SWCNT produces a universal, facile and cost-effective sensing platform for lead ions. The concept can be applied to the design of detection assays for other metal ions or small molecules.     

金属离子对氯过氧化物酶在25℃到55℃激活作用以及机理的研究

基于氯过氧化物酶（CPO）的卤化活性分析,发现某些碱土金属（Ca2+, Mg2+）和过渡金属（Co2+, Ni2+）对CPO具有明显的激活及稳定化作用。例如25 ºC时与CPO在纯缓冲溶液中相比,在75 μmol·L-1 Ca2+,90 μmol·L-1 Mg2+,90 μmol·L-1 Ni2+及105 μmol·L-1 Co2+存在时CPO可分别获得1.33,1.37,1.34 及1.27倍的最大相对活性。而在55 ºC,没有金属离子存在时,CPO 30分钟后仅能保留40%的活性,但在Ca2+,Mg2+离子的介质中,CPO的活性可分别保留81% 和 75%。推测这是由于金属离子结合在CPO活性中心周围的酸-碱催化位点Glu183, His105 and Asp106上,通过底物浓集和诱导有利构象来激活CPO. 同时动力学研究表明金属离子对CPO的激活归因于催化效率（kcat）的提高,以及CPO对底物亲和性及选择性的改善。