DNAzyme-Controlled Cleavage of Dimer and Trimer Origami Tiles

Dimers of origami tiles are bridged by the Pb²⁺-dependent DNAzyme sequence and its substrate or by the histidine-dependent DNAzyme sequence and its substrate to yield the dimers T₁–T₂ and T₃–T₄, respectively. The dimers are cleaved to monomer tiles in the presence of Pb²⁺-ions or histidine as triggers. Similarly, trimers of origami tiles are constructed by bridging the tiles with the Pb²⁺-ion-dependent DNAzyme sequence and the histidine-dependent DNAzyme sequence and their substrates yielding the trimer T₁–T₅–T₄. In the presence of Pb²⁺-ions and/or histidine as triggers, the programmed cleavage of trimer proceeds. Using Pb²⁺ or histidine as trigger cleaves the trimer to yield T₅–T₄ and T₁ or the dimer T₁–T₅ and T₄, respectively. In the presence of Pb²⁺-ions and histidine as triggers, the cleavage products are the monomer tiles T₁, T₅, and T₄. The different cleavage products are identified by labeling the tiles with 0, 1, or 2 streptavidin labels and AFM imaging

Hemin/G-Quadruplex-Catalyzed Aerobic Oxidation of Thiols to Disulfides: Application of the Process for the Development of Sensors and Aptasensors and for Probing Acetylcholine Esterase Activity

This study describes the novel hemin/G-quadruplex DNAzyme-catalyzed aerobic oxidation of thiols to disulfides and the respective mechanism. The mechanism of the reaction involves the DNAzyme-catalyzed oxidation of thiols to disulfides and the thiol-mediated autocatalytic generation of H₂O₂ from oxygen. The coupling of a concomitant H₂O₂-mediated hemin/G-quadruplex-catalyzed oxidation of Amplex Red to the fluorescent resorufin as a transduction module provides a fluorescent signal for probing the catalyzed oxidation of the thiol to disulfides and for probing sensing processes that yield the hemin/G-quadruplex as a functional label. Accordingly, a versatile sensing method for analyzing thiols (l-cysteine, glutathione) using the H₂O₂-mediated DNAzyme-catalyzed oxidation of Amplex Red to the resorufin was developed. Also, the l-cysteine and Amplex Red system was implemented as an auxiliary fluorescent transduction module for probing recognition events that form the catalytic hemin/G-quadruplex structures. This is exemplified with the development of thrombin aptasensor. The thrombin/thrombin binding aptamer recognition complex binds hemin, and the resulting catalytic complex activates the auxiliary transduction module, involving the aerobic oxidation of l-cysteine and the concomitant formation of the fluorescent resorufin. Finally, the hemin/G-quadruplex DNAzyme/Amplex Red system was used to follow the activity of acetylcholine esterase, AChE, and to probe its inhibition. The AChE-catalyzed hydrolysis of acetylthiocholine to the thiol-functionalized thiocholine enabled the probing of the enzymatic activity of AChE through the hemin/G-quadruplex-catalyzed aerobic oxidation of thiocholine to the respective disulfide and the concomitant generation of the fluorescent resorufin product

Enzyme-Free Amplified Detection of DNA by an Autonomous Ligation DNAzyme Machinery

The Zn²⁺-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal autocatalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10^–11 M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis

Zn²⁺-Ligation DNAzyme-Driven Enzymatic and Nonenzymatic Cascades for the Amplified Detection of DNA

A generic fluorescence sensing platform for analyzing DNA by the Zn²⁺-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a “helper” nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components (“helper” sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn²⁺-dependent ligation DNAzyme-driven activation of the Mg²⁺-dependent DNAzyme. According to this method, the Mg²⁺-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay–Sachs genetic disorder mutant

Switchable Catalytic DNA Catenanes

Two-ring interlocked DNA catenanes are synthesized and characterized. The supramolecular catenanes show switchable cyclic catalytic properties. In one system, the catenane structure is switched between a hemin/G-quadruplex catalytic structure and a catalytically inactive state. In the second catenane structure the catenane is switched between a catalytically active Mg²⁺-dependent DNAzyme-containing catenane and an inactive catenane state. In the third system, the interlocked catenane structure is switched between two distinct catalytic structures that include the Mg²⁺- and the Zn²⁺-dependent DNAzymes

Amplified Surface Plasmon Resonance and Electrochemical Detection of Pb²⁺ Ions Using the Pb²⁺-Dependent DNAzyme and Hemin/G-Quadruplex as a Label

The hemin/G-quadruplex nanostructure and the Pb²⁺-dependent DNAzyme are implemented to develop sensitive surface plasmon resonance (SPR) and electrochemical sensing platforms for Pb²⁺ ions. A complex consisting of the Pb²⁺-dependent DNAzyme sequence and a ribonuclease-containing nucleic acid sequence (corresponding to the substrate of the DNAzyme) linked to a G-rich domain, which is “caged” in the complex structure, is assembled on Au-coated glass surfaces or Au electrodes. In the presence of Pb²⁺ ions, the Pb²⁺-dependent DNAzyme cleaves the substrate, leading to the separation of the complex and to the self-assembly of the hemin/G-quadruplex on the Au support. In one sensing platform, the Pb²⁺ ions are analyzed by following the dielectric changes at the surface as a result of the formation of the hemin/G-quadruplex label using SPR. This sensing platform is further amplified by the immobilization of the sensing complex on Au NPs (13 nm) and using the electronic coupling between the NPs and the surface plasmon wave as an amplification mechanism. This method enables the sensing of Pb²⁺ ions with a detection limit that corresponds to 5 fM. The second sensing platform implements the resulting hemin/G-quadruplex as an electrocatalytic label that catalyzes the electrochemical reduction of H₂O₂. This method enables the detection of Pb²⁺ with a detection limit of 1 pM. Both sensing platforms reveal selectivity toward the detection of Pb²⁺ ions

Autonomous Replication of Nucleic Acids by Polymerization/Nicking Enzyme/DNAzyme Cascades for the Amplified Detection of DNA and the Aptamer–Cocaine Complex

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer–substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg²⁺-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg²⁺-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM)

Amplified Detection of DNA through the Enzyme-Free Autonomous Assembly of Hemin/G-Quadruplex DNAzyme Nanowires

An enzyme-free amplified detection platform is described using the horseradish peroxidase (HRP)-mimicking DNAzyme as an amplifying label. Two hairpin structures that include three-fourths and one-fourth of the HRP-mimicking DNAzyme in caged, inactive configurations are used as functional elements for the amplified detection of the target DNA. In the presence of the analyte DNA, one of the hairpins is opened, and this triggers the autonomous cross-opening of the two hairpins using the strand displacement principle. This leads to the formation of nanowires consisting of the HRP-mimicking DNAzyme. The resulting DNA nanowires act as catalytic labels for the colorimetric or chemiluminescent readout of the sensing processes (the term “enzyme-free” refers to a protein-free catalyst). The analytical platform allows the sensing of the analyte DNA with a detection limit corresponding to 1 × 10^–13 M. The optimized system acts as a versatile sensing platform, and by coaddition of a “helper” hairpin structure any DNA sequence may be analyzed by the system. This is exemplified with the detection of the BRCA1 oncogene with a detection limit of 1 × 10^–13 M

Multiplexed Analysis of Genes and of Metal Ions Using Enzyme/DNAzyme Amplification Machineries

The progressive development of amplified DNA sensors using nucleic acid-based machineries, involving the isothermal autonomous synthesis of the Mg²⁺-dependent DNAzyme, is used for the amplified, multiplexed analysis of genes (Smallpox, TP53) and metal ions (Ag⁺, Hg²⁺). The DNA sensing machineries are based on the assembly of two sensing modules consisting of two nucleic acid scaffolds that include recognition sites for the two genes and replication tracks that yield the nicking domains for Nt.BbvCI and two different Mg²⁺-dependent DNAzyme sequences. In the presence of any of the genes or the genes together, their binding to the respective recognition sequences triggers the nicking/polymerization machineries, leading to the synthesis of two different Mg²⁺-dependent DNAzyme sequences. The cleavage of two different fluorophore/quencher-modified substrates by the respective DNAzymes leads to the fluorescence of F₁ and/or F₂ as readout signals for the detection of the genes. The detection limits for analyzing the Smallpox and TP53 genes correspond to 0.1 nM. Similarly, two different nucleic acid scaffolds that include Ag⁺-ions or Hg²⁺-ions recognition sequences and the replication tracks that yield the Nt.BbvCI nicking domains and the respective Mg²⁺-dependent DNAzyme sequences are implemented as nicking/replication machineries for the amplified, multiplexed analysis of the two ions, with detection limits corresponding to 1 nM. The ions sensing modules reveal selectivities dominated by the respective recognition sequences associated with the scaffolds

Application of DNA Machineries for the Barcode Patterned Detection of Genes or Proteins

The study introduces an analytical platform for the detection of genes or aptamer-ligand complexes by nucleic acid barcode patterns generated by DNA machineries. The DNA machineries consist of nucleic acid scaffolds that include specific recognition sites for the different genes or aptamer-ligand analytes. The binding of the analytes to the scaffolds initiate, in the presence of the nucleotide mixture, a cyclic polymerization/nicking machinery that yields displaced strands of variable lengths. The electrophoretic separation of the resulting strands provides barcode patterns for the specific detection of the different analytes. Mixtures of DNA machineries that yield, upon sensing of different genes (or aptamer ligands), one-, two-, or three-band barcode patterns are described. The combination of nucleic acid scaffolds acting, in the presence of polymerase/nicking enzyme and nucleotide mixture, as DNA machineries, that generate multiband barcode patterns provide an analytical platform for the detection of an individual gene out of many possible genes. The diversity of genes (or other analytes) that can be analyzed by the DNA machineries and the barcode patterned imaging is given by the Pascal’s triangle. As a proof-of-concept, the detection of one of six genes, that is, TP53, Werner syndrome, Tay-Sachs normal gene, BRCA1, Tay-Sachs mutant gene, and cystic fibrosis disorder gene by six two-band barcode patterns is demonstrated. The advantages and limitations of the detection of analytes by polymerase/nicking DNA machineries that yield barcode patterns as imaging readout signals are discussed