Joanna Kosman

Peroxidase-mimicking DNAzymes for biosensing applications: a review.

DNAzymes are single stranded DNA molecules that exhibit catalytic activity and are exploited in medicine, biology and material sciences. Development in this area is related to the many advantages of DNAzymes over conventional protein enzymes, such as thermal stability and simpler preparation. DNAzymes with peroxidase-like activity have recently attracted great interest. To assure such catalytic activity, oligonucleotides have to adopt a G-quadruplex structure, which can bind the hemin molecule. This system facilitates a redox reaction between the target molecule and hydrogen peroxide, which results in the appearance of an oxidized target molecule (product). DNAzymes with peroxidase-mimicking activity have great potential in bioanalytical chemistry. This review presents fundamentals concerning the design and engineering of DNAzymes with peroxidase-like activity, describes their properties and spectral characteristics and shows how DNAzymes can contribute to bioanalytical research. Examples of bioanalytical applications of DNAzymes with peroxidase-like activity include nucleic acid probes with DNAzyme labels for the detection of specific DNA sequences in colorimetric or chemiluminescent assays. Assays for telomerase or methyltransferase activity, which are potential targets in anticancer therapy, are also described in this review. Other applications include the determination of metal cations such as Ag(+), K(+), Hg(2+), Pb(2+) or Cu(2+) and amplified detection of small molecules such as adenosine, cocaine or AMP and proteins such as lysozyme or thrombin. In the last decade, DNAzymes have become part of numerous applications in many areas of science from chemistry to biology to medicine.

Optimization study of the catalytic activity of DNAzymes based on telomeric G-quadruplexes

AbstractOptimization studies of the procedure for peroxidase activity measurements with DNAzymes based on telomeric sequences and colorimetric indicator reactions are reported. Effect of metal cation, nature and concentration of surfactant, as well as thermal treatment of G-quadruplex sample are investigated. Nature of metal cation exhibited modest influence on the system performance. Great improvement of enzymatic activity of the telomeric quadruplexes in the presence of Brij 58 surfactant was observed. Further improvement of catalytic activity of the system based on human telomeric sequence was attained by applying a thermal treatment (heating/rapid cooling) procedure to prepare G-quadruplex/hemin complexes.

Conjugation of hemin to G-quadruplex forming oligonucleotide using click chemistry

Peroxidase-mimicking DNAzyme is one of the systems that recently gained a great interest. It has been successfully applied for designing numerous bioassays. The success of this system is connected to its advantages over a protein enzyme, horseradish peroxidase. Promising strategy for further improvement of performance of DNAzyme with peroxidase-like activity was proposed recently. It was based on the covalent attachment of hemin moiety to the G-quadruplex scaffold. We report here the first attempt of conjugating hemin to the G-quadruplex DNA using click chemistry approach. We modified hemin molecule through attachment of an azide-terminated linker to the porphyrin carboxylic group. Two click chemistry approaches were examined to conjugate the azide-modified hemin to a G-quadruplex oligonucleotide: copper-catalyzed and Cu-free cycloaddition reactions. Using Cu-free click reaction, we successfully synthesized G-quadruplex-hemin conjugate that exhibited promising peroxidase activity.

Dataset on characterization of hemin-azide derivative and DNA oligonucleotide-hemin conjugate

In this article newly synthesized azide derivative of hemin and DNA-hemin conjugate are characterized. Hemin-azide was purified using HPLC and characterized using elemental analysis, IR and NMR. The DNA-hemin conjugate was obtained via click chemistry [1] and click reaction was carried out using traditional Cu-catalyzed and Cu-free approaches. The final product was successfully obtained using Cu-free cycloaddition. The identity of product was confirmed using Maldi TOF spectrometry. Obtained hemin-DNA conjugate exhibited peroxidase-like activity.

FRET study of G-quadruplex forming fluorescent oligonucleotide probes at the lipid monolayer interface.

Spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes at the cationic dioctadecyldimethylammonium bromide (DODAB) monolayer interface are reported. Two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence, were end-labeled with fluorescent groups (FAM and TAMRA) to give FRET probes F19T and F21T, respectively. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na(+) and K(+)). Fluorescence spectra of G-quadruplex FRET probes at the monolayer interface are reported for the first time. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. The effect of the presence of DODAB monolayer, metal cations and the surface pressure of monolayer on spectral behavior of FRET probes were examined. Adsorption of probe at the cationic monolayer interface resulted in the FRET signal enhancement even in the absence of metal cations. Variation in the monolayer surface pressure exerted rather modest effect on the spectral properties of probes. The fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the monolayer interface.

Bioactive papaverine derivatives bind G-quadruplexes selectively

G-quadruplexes are a family of DNA secondary structures resulting from the folding of a guanine-rich sequence. Targeting quadruplexes by small molecules is an approach that is currently being studied with the aim of exploring their biological roles and developing new anti-cancer agents. There is evidence that the formation of G4 structures by telomeric DNA can be used to inhibit the enzyme activity of telomerase, and thereby to activate the pathway to senescence in tumour cells. It was previously shown that the papaverine oxidation products 6a,12a-diazadibenzo-[a,g]fluorenylium derivative (ligand I) and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium chloride (ligand II) bind to G-quadruplex representing the human telomeric sequence. These ligands possess the ability to inhibit telomerase and polymerase action at the micromolar level. Here we report a DNA binding study on these two ligands and a new derivative 2-(2-carboxy-4,5-dimethoxyphenyl0-6,7-dimethoxyisoquiloliniuminner salt (ligand III) in order to evaluate their binding selectivity to samples of nucleic acids (ssDNA, dsDNA, triplexes, and quadruplexes). Simultaneous investigations on several DNA-ligand complexes carried out using an equilibrium dialysis approach revealed pronounced binding selectivity of ligand I and ligand II to tetraplex DNA structures over the doublestranded DNA forms.

A New Strategy for Silver Deposition on Au Nanoparticles with the Use of Peroxidase-Mimicking DNAzyme Monitored via a Localized Surface Plasmon Resonance Technique

Peroxidase-mimicking DNAzyme was applied as a catalyst of silver deposition on gold nanoparticles. This DNAzyme is formed when hemin binds to the G-quadruplex-forming DNA sequence. Such a system is able to catalyze a redox reaction with a one- or two-electron transfer. The process of silver deposition was monitored via a localized surface plasmon resonance technique (LSPR), which allows one to record scattering spectrum of a single nanoparticle. Our study showed that DNAzyme is able to catalyze silver deposition. The AFM experiments proved that DNAzyme induced the deposition of silver shells of approximately 20 nm thickness on Au nanoparticles (AuNPs). Such an effect is not observed when hemin is absent in the system. However, we noticed non-specific binding of hemin to the capture oligonucleotides on a gold NP probe that also induced some silver deposition, even though the capture probe was unable to form G-quadruplex. Analysis of SEM images indicated that the surface morphology of the silver layer deposited by DNAzyme is different from that obtained for hemin alone. The proposed strategy of silver layer synthesis on gold nanoparticles catalyzed by DNAzyme is an innovative approach and can be applied in bioanalysis (LSPR, electrochemistry) as well as in material sciences.

Comparison of Characteristics and DNAzyme Activity of G4–Hemin Conjugates Obtained via Two Hemin Attachment Methods

Two conjugation methods using different linkers were applied for the investigation of the spectral characteristics and activity of G-quadruplex (G4)–hemin conjugates. For this purpose, two G-quadruplex-forming DNA sequences were selected, and then conjugated to a hemin molecule via either amine coupling or a click reaction. The products obtained via these two methods differed in their chemistry and the length of the linker between the DNA and hemin molecules. Spectral characteristics revealed that both methods produced conjugates that were more thermally stable than G4/hemin complexes. Despite similar spectral characteristics, the conjugates obtained via these two methods differed in their DNAzyme activity. G4–hemin conjugates obtained through amine coupling exhibited higher activity than conjugates obtained through a click reaction. This was potentially due to the length and chemistry of the linker, which was 30 atoms long following the click reaction, but only six atoms long following amine coupling. A longer connector favors higher flexibility, and hence, reduces the binding of hemin with G4. The aromatic groups present in the linker obtained through the click reaction can also disturb the G4–hemin interaction. However, the conjugation of G4 DNA to hemin via the click reaction was connected to a higher yield, and did not require any sophisticated synthesis equipment.

Catalytic G-quadruplexes for the Detection of Telomerase Activity

The unique properties of G4-based nucleic acids provide the base for a variety of applications.A whole set of biomedical as well as bioanalytical applications is based on the ability of G-quartets to stabilize defined three-dimensional nucleic acid structures that exhibit a high affinity to a target molecule. Such aptamers can therefore show similar binding properties as antibodies with comparable applications in diagnostics and therapy, but exhibit striking advantages like ex-vivo synthesis and increased physicochemical stability. Moreover, they can even show catalytic behavior that can be utilized for bioanalytical purposes. The chapter contains examples for applications in these fields.The structural properties described in previous chapters are the base for applications in molecular nanotechnology and –electronics. Nucleic acids represent the most promising materials in these fields, and here G4 structures show even outstanding mechanical stability and length control from the nano- into the micrometer range. An important step on the way to respective applications is the integration of G4-based nanostructures into technical environments such as microelectrodes. Here electrical field-based approaches – e.g. dielectrophoresis DEP – represent the most promising technique, which has been demonstrated for the integration and subsequent characterization of even single G4 structures. These techniques have also been used to enable the characterization of electrical properties of G4 assemblies as described in this chapter.In conclusion, by presenting various biomedical as well as nanobiotechnological demonstrations this chapter demonstrates the great application potential of G4-based nanostructures.