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

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

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

Self-Assembly of Luminescent Ag Nanocluster-Functionalized Nanowires

Two different methods to self-assemble red- or yellow-luminescent nucleic acids-stabilized Ag nanoclusters (NCs) nanowires are presented. By one method, the autonomous hybridization–polymerization process between two nucleic acids leads to polymer chains consisting of sequence-specific loops for the stabilization of the red- or yellow-emitting Ag NCs. By the other method, the nucleic acid-triggered hybridization chain reaction (HCR) involving the cross-opening of two functional hairpins leads to sequence-specific DNA loops and a nucleic acid scaffold that stabilize the respective red- or yellow-emitting Ag NCs. The micrometer-long luminescent Ag NC-functionalized nanowires are imaged by AFM and confocal microscopy

Amplified Analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires

A systematic study of the amplified optical detection of DNA by Mg²⁺-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10^–9 M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg²⁺-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10^–14 M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene

Gossypol-Cross-Linked Boronic Acid-Modified Hydrogels: A Functional Matrix for the Controlled Release of an Anticancer Drug

Anticancer drug gossypol cross-links phenylboronic acid-modified acrylamide copolymer chains to form a hydrogel matrix. The hydrogel is dissociated in an acidic environment (pH 4.5), and its dissociation is enhanced in the presence of lactic acid (an α-hydroxy carboxylic acid) as compared to formic acid. The enhanced dissociation of the hydrogel by lactic acid is attributed to the effective separation of the boronate ester bridging groups through the formation of a stabilized complex between the boronic acid substituent and the lactic acid. Because lactic acid exists in cancer cells in elevated amounts and the cancer cells’ environment is acidic, the cross-linked hydrogel represents a stimuli-responsive matrix for the controlled release of gossypol. The functionality is demonstrated and characterized by rheology and other spectroscopic means

Switchable Catalytic Acrylamide Hydrogels Cross-Linked by Hemin/G-Quadruplexes

Copolymer chains consisting of acrylamide units and guanine (G)-containing oligonucleotide-tethered acrylamide units undergo, in the presence of K⁺ ions, cross-linking by G-quadruplexes to yield a hydrogel. The hydrogel is dissociated upon addition of 18-crown-6 ether that traps the K⁺ ions. Reversible formation and dissociation of the hydrogel is demonstrated by the cyclic addition of K⁺ ions and 18-crown-6 ether, respectively. Formation of the hydrogel in the presence of hemin results in a hemin/G-quadruplex-cross-linked catalytic hydrogel mimicking the function of horseradish peroxidase, reflected by the catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), ABTS^2–, by H₂O₂ to ABTS^.– and by the catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂. Cyclic “ON” and “OFF” activation of the catalytic functions of the hydrogel are demonstrated upon the formation of the hydrogel in the presence of K⁺ ions and its dissociation by 18-crown-6 ether, respectively. The hydrogel is characterized by rheology measurements, circular dichroism, and probing its chemical and photophysical properties