Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase

A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.     

DNAzyme-based turn-on chemiluminescence assays in homogenous media

In this work, novel biosensing systems were developed for DNAzyme-based assays in homogenous aqueous media. The two halves of a horseradish peroxidase mimicking DNAzyme were assembled onto different gold nanoparticle surfaces through hybridization with corresponding linking DNA sequences. In the analyses, the target molecules were recognized by the linking DNA. This recognition broke the hybridization and released the DNAzyme halves from the nanoparticle surface into the solution. Together, both the DNAzyme halves combined with a cofactor hemin and turned into a catalytic hemin/G-quadruplex structure, which amplified the luminol oxidation for a turn-on chemiluminescence signaling. Based on this nanoparticle-based DNAzyme-halves design, only low background noise showed up within the homogenous solution and no separation was required in the detection steps. Aptasensor and DNA sensor were developed and analyses of the target molecules adenosine and target DNA were achieved down to 0.7 μM and 0.3 nM respectively with satisfactory selectivity.     

Comparison of photovoltaic behaviors for horseradish peroxidase and its mimicry by surface photovoltage spectroscopy

Surface photovoltage spectroscopy (SPS) was chosen to study the photovoltaic behavior of horseradish peroxidase (HRP), hemin and immobilized hemin (poly(NIPAAm/MBA/hemin)). Different photovoltaic behaviors were observed in these three systems. In air, similar SPS curves were found for HRP and poly(NIPAAm/MBA/hemin) with different response intensities. However, poly(NIPAAm/MBA/hemin) showed a wider changing range upon increasing the positive and negative bias to 1.0 V. The SPS of hemin showed a total different behavior when an external positive potential was applied. In vacuum, clearly different photovoltaic behaviors were found. Moreover, the response value decreased when HRP was exposed to O2, the SPS intensity was different from that in air, and could be altered by changing the external biases. On the other hand, the SPS could not be changed before and after poly(NIPAAm/MBA/hemin) was exposed to O2. These differences may result from different chemical microenvironments for hemin in HRP versus that in poly(NIPAAm/MBA/hemin). It could be concluded that H2O and O2 were important factors affecting the photovoltage response in HRP, but only H2O played this important role in poly(NIPAAm/MBA/hemin).     

Studies of the activity of peroxidase-like DNAzyme by modifying 3'- or 5'-end of aptamers

In the presence of hemin and under appropriate conditions, some modalities of G-quadruplexes can form a peroxidase-like DNAzyme that has been widely used in biology. Structure-function studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G-quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G-quadruplex sequences by adding different lengths of oligo-dT to the 3'- or 5'-end of the aptamers. The results suggested that adding dT(n) to the 5'-end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dT(n) to the 3'-end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H(2) O(2)), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.     

Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity

G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3′ end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3′ adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid–base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions.     

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

The effects of various refolding additives, including metal cofactors, organic co‐solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well‐known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion‐containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca²⁺ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3‐fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea‐containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor‐containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.     

Studies of the Activity of Peroxidase‐Like DNAzyme by Modifying 3′‐ or 5′‐End of Aptamers

In the presence of hemin and under appropriate conditions, some modalities of G‐quadruplexes can form a peroxidase‐like DNAzyme that has been widely used in biology. Structure? function studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G‐quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G‐quadruplex sequences by adding different lengths of oligo‐dT to the 3′‐ or 5′‐end of the aptamers. The results suggested that adding dT_n to the 5′‐end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dT_n to the 3′‐end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H₂O₂), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.     

Microflow injection potassium bioassay based on G‐quadruplex DNAzyme‐enhanced chemiluminescence

By taking advantage of microflow injection chemiluminescence analysis, we developed a distinctive microfluidic bioassay method based on G‐Quadruplex DNAzyme‐enhanced chemiluminescence for the determination of K⁺ in human serum. AGRO100, the G‐rich oligonucleotide with high hemin binding affinity was primarily selected as a K⁺ recognition element. In the presence of K⁺, AGRO100 folded into G‐quadruplex and bound hemin to form DNAzyme, which catalyzed the oxidation of luminol by H₂O₂ to produce chemiluminescence. The intensity of chemiluminescence increased with the K⁺ concentration. In the study, the DNAzyme showed both long‐term stability and high catalytic activity; other common cations at their physiological concentration did not cause notable interference. With only 6.7 × 10^?13 mol of AGRO100 consumption per sample, a linear response of K⁺ ranged from 1 to 300 µmol/L, the concentration detection limit 0.69 µmol/L (S/N = 3) and the absolute detection limit 1.38 × 10^?12 mol were obtained. The precision of 10 replicate measurements of 60 µmol/L K⁺ was found to be 1.72% (relative standard deviation). The accuracy of the method was demonstrated by analyzing real human serum samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Label-free fluorescence method for screening G-quadruplex ligands

G-quadruplex ligands can interfere with the telomere structure, telomere elongation/replication, and proliferation of cancer cells. A key element in the development of potent G-quadruplex ligands is the screening of large chemical libraries of potential candidates. Here, we describe a simple fluorescence method for screening of G-quadruplex ligands. The method is based on the ability of G-quadruplex ligands to displace hemin from G-quadruplex-based DNAzyme, resulting in a decrease of its catalytic activity on the fluorescence-developing reaction between p-hydroxyphenylacetic acid and H(2)O(2). The method eliminates the requirement for expensive and time-consuming preparation of labeled DNA. Our method provides a simple, cheap, and sensitive approach to screen G-quadruplex ligands (potential antitumor drugs).     

Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.     

Homology modeling and molecular dynamics study of West Nile virus NS3 protease: a molecular basis for the catalytic activity increased by the NS2B cofactor

The West Nile virus (WNV) NS3 serine protease, which plays an important role in assembly of infective virion, is an attractive target for anti-WNV drug development. Cofactors NS2B and NS4A increase the catalytic activity of NS3 in dengue virus and Hepatitis C virus, respectively. Recent studies on the WNV-NS3 characterize the catalytically active form of NS3 by tethering the 40-residue cofactor NS2B. It is suggested that NS2B is essential for the NS3 activity in WNV, while there is no information of the WNV-NS3-related crystal structure. To understand the role of NS2B/substrate in the NS3 catalytic activity, we built a series of models: WNV-NS3 and WNV-NS3-NS2B and WNV-NS3-NS2B-substrate using homology modeling and molecular modeling techniques. Molecular dynamics (MD) simulations were performed for 2.75 ns on each model, to investigate the structural stabilization and catalytic triad motion of the WNV NS3 protease with and without NS2B/substrate. The simulations show that the NS3 rearrangement occurs upon the NS2B binding, resulting in the stable D75-OD1...H51-NH hydrogen bonding. After the substrate binds to the NS3-NS2B active site, the NS3 protease becomes more stable, and the catalytic triad is formed. These results provide a structural basis for the activation and stabilization of the enzyme by its cofactor and substrate.     

Thermostability, solvent tolerance, catalytic activity and conformation of cofactor modified horseradish peroxidase

Artificial prosthetic groups, HeminD1 and HeminD2, were designed and synthesized, which contain one benzene ring and one carboxylic group or two carboxylic groups at the terminal of each propionate side chain of hemin, respectively. HeminD1 and HeminD2 were reconstituted with apo-HRP successfully to produce the two novel HRPs, rHRP1 and rHRP2, respectively. The thermal and solvent tolerances of native and reconstituted HRPs were compared. The cofactor modification increased the thermostability both in aqueous buffer and some organic solvents, and also enhanced the tolerance of some organic solvents. To determine the conformation stability, the unfolding of native and reconstituted HRPs by heat was investigated. T_m was increased from 70.0 °C of nHRP to 75.4 °C of rHRP1 and 76.5 °C of rHRP2 after cofactor modification. Kinetic studies indicated that the cofactor modification increased the substrate affinity and catalytic efficiency both in aqueous buffer and some organic solvents. The catalytic efficiency for phenol oxidation was increased by 55% for rHRP1 in aqueous buffer, and it was also increased by 70% for rHRP1 in 10% ACN. Spectroscopic studies proved that the cofactor modification changed the microenvironment of both heme and tryptophan, increased α-helix content, and increased the tertiary structure around the aromatic residue in HRP. The improvements of catalytic properties are related to these changes of the conformation. The introduction of the hydrophobic domain as well as the retention of the moderate carboxylic group in active site is an efficient method to improve the thermodynamic and catalytic efficiency of HRP.     

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100 mM K⁺, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. ¹D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.     

Characterization of the G‐quadruplex structure of a catalytic DNA with peroxidase activity

It has been reported that the complexes formed by hemin and some G‐quadruplexes can be developed as a new class of DNAzyme with peroxidase activity. This kind of DNAzyme has received a great deal of attention. But to date, the actual G‐quadruplex structure that can provide hemin with enhanced peroxidase activity is in doubt. Herein, the G‐quadruplex structure of CatG4, a 21‐nucleotide DNA oligomer which was previously reported to bind hemin and the resulting complex exhibiting enhanced peroxidase activity, was characterized by fluorescence and circular dichroism measurements. The results suggest that the catalytically active form of CatG4 may be a unimolecular parallel quadruplex rather than a unimolecular chair‐type antiparallel quadruplex or a multistranded parallel quadruplex. In addition, the fluorescence analysis of labeled oligonucleotides may be developed as a supplementary tool for the study of DNA conformations. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 331–339, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mechanism of low-density lipoprotein oxidation by hemoglobin-derived iron

Excellular hemoglobin is an extremely active oxidant of low-density lipoproteins (LDL), a phenomenon explained so far by different mechanisms. In this study, we analyzed the mechanism of met-hemoglobin oxidability by comparing its mode of operation with other hemoproteins, met-myoglobin and horseradish peroxidase (HRP) or with free hemin. The kinetics of met-hemoglobin activity toward LDL lipids and protein differed from that of met-myoglobin and HRP, both quantitatively and qualitatively. Those differences were further clarified by analyzing heme transfer from the above-mentioned hemoproteins to LDL. It appeared that met-hemoglobin transferred most of its hemin to LDL, and the presence of H(2)O(2) accelerated the process. In contrast, met-myoglobin partially released hemin, but only in the presence of H(2)O(2), while HRP could not transfer heme at all. The minor amount of hemin transferred from met-myoglobin to LDL sufficed to trigger ApoB oxidation, forming covalent aggregates via inter-bityrosines. This indicated that heme bound to high affinity site(s) is responsible for oxidation. LDL components providing the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra revealed that the high affinity site of monomeric hemin is located on the LDL protein, ApoB. The complex heme-CO-ApoB underwent instantaneous oxidation to hemin-ApoB, and the bound hemin then slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented LDL oxidation by trapping hemoprotein transferable heme. We concluded that met-hemoglobin exerts its oxidative activity on LDL via transfer of heme, which serves as a vehicle for iron insertion into the LDL protein, leading to formation of atherogenic LDL aggregates.     

Correct assembly of iron-sulfur cluster FS0 into Escherichia coli dimethyl sulfoxide reductase (DmsABC) is a prerequisite for molybdenum cofactor insertion

The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = 3/2) in its reduced form, resulting in an EPR spectrum with a peak at g ～ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of dimethyl sulfoxide reduction. Comparison with nitrate reductase A (NarGHI) indicates that a sequence of residues ((18)CTVNC(22)) plays a role in both FS0 and Mo-bisPGD coordination. A DmsA(ΔN21) mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from (18)C(A)TVNC(B)GSRC(C)P(27) to (18)C(A)TYC(B)GVGC(C)G(26) (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsA(R61K) mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.     

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.     

Highly effective colorimetric and visual detection of ATP by a DNAzyme-aptamer sensor

A new and simple method was developed to detect adenosine triphosphate (ATP) by using a DNAzyme aptamer sensor. The DNAzyme used was a single‐stranded DNA that could combine with hemin. The aptamer, a single, short nucleic acid sequence that can specifically bind with many targets, was an anti‐ATP aptamer. Two DNA sequences were designed: i) a functional chain (Chain A) consisting of two parts, i.e., the anti‐ATP aptamer (recognition part) and the DNAzyme (signal transduction part) and ii) a blocker chain (Chain B), which could partially hybridize with Chain A. The hybridized chains A and B were unfolded by the addition of ATP and hemin, and the blocker chain and the complex of the functional chain with ATP and hemin were in solution. The DNAzyme in the functional chain formed a G‐quadruplex with hemin and then catalyzed the oxidation by H₂O₂ of 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS²⁻) to the colored ABTS^.− radical. The color change caused by this reaction could be clearly observed by naked eye, and the absorbance was recorded at 414 nm. The detection limit was 1×10⁻⁶ M .     

基于G4-配体的DNA模拟酶在生物传感中的发展与应用

鸟苷酸-四联体DNA (G4 DNA)是由富含串联重复鸟苷酸(G)的DNA或RNA序列形成的G4片层,并堆叠而成一类独特的核酸二级结构。G4 DNA结合多种特异性配体可形成具有催化过氧化氢活性的G4 DNA模拟酶(G4 DNAzyme)。由于G4 DNAzyme存在着序列构成简便灵活、适合多样传感平台检测等特点,其在新型生物传感方法研发、医学检测新技术研究等领域中应用前景广阔。本文主要依据G4 DNA结合配体的不同,对近年来新发展出的G4 DNAzyme进行分类与回顾,归纳为含氯化血红素(hemin)的G4-Hemin DNAzyme与非G4-Hemin DNAzyme。前者是目前G4 DNA模拟酶的研究主流——本文主要归纳了G4-Hemin DNAzyme在金属阳离子、生物小分子及生物大分子的检测分析方向上所取得的重要研究进展,并阐述其在生物传感领域的影响和优势;后者中的配体则主要包括金属阳离子N-甲基吗啡啉(4-methylmorpholine, NMM)、硫磺素T(thioflavin T,ThT)及新型金属配体(Cu²⁺Ce³⁺)等。...     

Metal ion as both a cofactor and a probe of metal-binding sites in a uranyl-specific DNAzyme: a uranyl photocleavage study

DNAzymes are known to bind metal ions specifically to carry out catalytic functions. Despite many studies since DNAzymes were discovered nearly two decades ago, the metal-binding sites in DNAzymes are not fully understood. Herein, we adopt uranyl photocleavage to probe specific uranyl-binding sites in the 39E DNAzyme with catalytically relevant concentrations of uranyl. The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand. Control experiments using two 39E DNAzyme mutants revealed a different cleavage pattern of the mutated region. Another DNAzyme, the 8–17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well. In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated. Together, these experiments suggest that uranyl photocleavage has been successfully used to probe catalytically relevant uranyl-binding sites in the 39E DNAzyme. As uranyl is the cofactor of the 39E DNAzyme as well as the probe, specific uranyl binding has now been identified without disruption of the structure.