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

pH-Programmable DNA Logic Arrays Powered by Modular DNAzyme Libraries

Nature performs complex information processing circuits, such the programmed transformations of versatile stem cells into targeted functional cells. Man-made molecular circuits are, however, unable to mimic such sophisticated biomachineries. To reach these goals, it is essential to construct programmable modular components that can be triggered by environmental stimuli to perform different logic circuits. We report on the unprecedented design of artificial pH-programmable DNA logic arrays, constructed by modular libraries of Mg²⁺- and UO₂²⁺-dependent DNAzyme subunits and their substrates. By the appropriate modular design of the DNA computation units, pH-programmable logic arrays of various complexities are realized, and the arrays can be erased, reused, and/or reprogrammed. Such systems may be implemented in the near future for nanomedical applications by pH-controlled regulation of cellular functions or may be used to control biotransformations stimulated by bacteria

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

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)

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

Smart Mesoporous SiO₂ Nanoparticles for the DNAzyme-Induced Multiplexed Release of Substrates

The fluorescent dyes methylene blue, MB⁺, and thionine, Th⁺, can be trapped in the pores of mesoporous silica, MP-SiO₂, by means of functional nanostructures consisting of the Mg²⁺- or Zn²⁺-dependent DNAzyme sequences. In the presence of Mg²⁺ or Zn²⁺ ions the respective DNAzymes are activated, leading to the specific cleavage of the respective caps, and the selective release of MB⁺ or Th⁺. The enlargement of the conserved loop domains of the Mg²⁺- or Zn²⁺-dependent DNAzyme sequences with foreign nucleotides prohibits the formation of active DNAzymes and eliminates the release of the respective dyes. This is due to the flexibility of the loops that lacks affinity for the association of the ions. The insertion of aptamer sequences (e.g., the adenosine-5′-triphosphate (ATP) aptamer) or ion-binding sequences (e.g., T-rich Hg²⁺ ion-binding domains) as foreign components to the loop regions allows the formation of active Mg²⁺- or Zn²⁺-dependent DNAzyme structures through the cooperative formation of aptamer-ATP complexes or T-Hg²⁺-T bridges. These aptamer–substrate complexes or T-Hg²⁺-T bridges allosterically stabilize and activate the DNAzymes, thus allowing the selective release of the fluorescent substrates MB⁺ or Th⁺. The metal ion-driven DNAzyme release of substrates from the pores of MP-SiO₂, and particularly the allosteric activation of the DNAzymes through cooperative aptamer–substrate complexes or metal-ion bridges, has important future nanomedical implications for targeted release of drugs. This is demonstrated with the triggered release of the anticancer drug, doxorubicin, by the Mg²⁺-DNAzyme-locked pores or by the aptamer-ATP complex-triggered activation of the Mg²⁺-dependent DNAzyme

Switching Photonic and Electrochemical Functions of a DNAzyme by DNA Machines

DNA nanostructures acting as DNA machines are described. Specifically, DNA “walkers” assembled on nucleic acid scaffolds and triggered by fuel/antifuel strands are activated in solution or on surfaces, for example, electrodes or semiconductor CdSe/ZnS quantum dots (QDs). The DNA machines led to the switchable formation or dissociation of the hemin/G-quadruplex DNAzyme on the DNA scaffolds. This enabled the chemiluminescence, chemiluminescence resonance energy transfer (CRET), electrochemical, or photoelectrochemical transduction of the switchable states of the different DNA machines

Evaluation of DNA Methyltransferase Activity and Inhibition via Isothermal Enzyme-Free Concatenated Hybridization Chain Reaction

Methyltransferase (MTase)-catalyzed DNA methylation plays a vital role in the biological epigenetic processes of key diseases and has attracted increasing attention, making the amplified detection of MTase activity of great significance in clinical disease diagnosis and treatment. Herein, we developed an isothermal, enzyme-free, and autonomous strategy for analyzing MTase activity based on concatenated hybridization chain reaction (C-HCR)-mediated Förster resonance energy transfer (FRET). In a typical C-HCR procedure without MTase (Dam), Y-shaped initiator DNA activates upstream HCR-1 to assemble a double-stranded DNA (dsDNA) copolymeric nanowire consisting of multiple tandem DNA trigger units that motivate downstream HCR-2 to successively bring a fluorophore donor/acceptor (FAM/TAMRA) pair into close proximity, leading to the generation of an amplified FRET readout signal. The target Dam MTase and auxiliary DpnI endonuclease can sequentially and specifically recognize/methylate and cleave the Y-shaped initiator oligonucleotide, respectively, and thus prohibit the C-HCR process and FRET signal generation, resulting in the construction of a signal-on sensing platform for MTase assay. Our proposed isothermal enzyme-free C-HCR amplification approach was further utilized for screening MTase inhibitors. Furthermore, the proposed C-HCR approach can be easily adapted for probing other different MTases and for screening the corresponding inhibitors just by changing the recognition sequence of Y-shaped initiator DNA through a “plug-and-play” format. It provides a versatile and robust tool for highly sensitive detection of various biotransformations and thus holds great promise in clinical assessment and diagnosis

Programmed DNAzyme-Triggered Dissolution of DNA-Based Hydrogels: Means for Controlled Release of Biocatalysts and for the Activation of Enzyme Cascades

Acrylamide/acrylamide-modified nucleic acid copolymer chains provide building units for the construction of acrylamide–DNA hydrogels. Three different hydrogels are prepared by the cross-linking of the acrylamide–DNA chains with metal ion-dependent DNAzyme sequences and their substrates. The metal ion-dependent DNAzyme sequences used in the study include the Cu²⁺-, Mg²⁺-, and Zn²⁺-dependent DNAzymes. In the presence of the respective metal ions, the substrates of the respective DNAzymes are cleaved, leading to the separation of the cross-linking units and to the dissolution of the hydrogel. The different hydrogels were loaded with a fluorophore-modified dextran or with a fluorophore-functionalized glucose oxidase. Treatment of the different hydrogels with the respective ions led to the release of the loaded dextran or the enzyme, and the rates of releasing of the loaded macromolecules followed the order of Cu²⁺ > Mg²⁺ > Zn²⁺. Also, the different hydrogels were loaded with the enzymes β-galactosidase (β-Gal), glucose oxidase (GOx), or horseradish peroxidase (HRP). In the presence of the appropriate metal ions, the respective hydrogels were dissolved, resulting in the activation of the β-Gal/GOx or GOx/HRP bienzyme cascades and of the β-Gal/GOx/HRP trienzyme cascade