Sequence Effect on the Activity of DNAzyme with Covalently Attached Hemin and Their Potential Bioanalytical Application.

In this work we investigated the effect of a DNA oligonucleotide sequence on the activity of a DNAzyme with covalently attached hemin. For this purpose, we synthesized seven DNA-hemin conjugates. All DNA-hemin conjugates as well as DNA/hemin complexes were characterized using circular dichroism, determination of melting temperatures and pKa of hemin. We observed that hemin conjugation in most cases led to the formation of parallel G-quadruplexes in the presence of potassium and increased thermal stability of all studied systems. Although the activity of DNA-hemin conjugates depended on the sequence used, the highest activity was observed for the DNA-hemin conjugate based on a human telomeric sequence. We used this DNAzyme for development of "sandwich" assay for detection of DNA sequence. For this assay, we used electric chip which could conduct electricity after silver deposition catalyzed by DNAzyme. This method was proved to be selective towards DNA oligonucleotides with mismatches and could be used for the detection of the target. To prove the versatility of our DNAzyme probe we also performed experiments with streptavidin-coated microplates. Our research proved that DNAzyme with covalently attached hemin can be used successfully in the development of heterogeneous assays.

Thrombin-Binding Aptamer with Inversion of Polarity Sites (IPS): Effect on DNAzyme Activity and Anticoagulant Properties.

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K+ or NH4+ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.

Light-Induced Oxidase Activity of DNAzyme-Modified Quantum Dots.

Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H2O2-free DNAzyme system with oxidase activity. Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH2) and carboxylated quantum dots (CdTe@COOH QDs). The obtained products were characterized by spectroscopic methods (UV-Vis, fluorescence, circular dichroizm (CD), and IR) and the transmission electron microscopy (TEM) technique. A QD-DNA system with a low polydispersity and high stability in aqueous solutions was successfully obtained. The catalytic activity of the QD-DNA conjugate was examined with Amplex Red and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) indicators using reactive oxygen species (ROS) generated by visible light irradiation. The synthesized QD-DNAzyme exhibited enhanced catalytic activity compared with the reference system (a mixture of QDs and DNAzyme). This proved the assumption that the covalent attachment of DNAzyme to the surface of QD resulted in a beneficial effect on its catalytic activity. The results proved that the QD-DNAzyme system can be used for generation of the signal by light irradiation. The light-induced oxidase activity of the conjugate was demonstrated, proving that the QD-DNAzyme system can be useful for the development of new cellular bioassays, e.g., for the determination of oxygen radical scavengers.

Development of fluorescence oligonucleotide probes based on cytosine- and guanine-rich sequences.

The properties of cytosine- and guanine-rich oligonucleotides contributed to employing them as sensing elements in various biosensors. In this paper, we report our current development of fluorescence oligonucleotide probes based on i-motif or G-quadruplex forming oligonucleotides for cellular measurements or bioimaging applications. Additionally, we also focus on the spectral properties of the new fluorescent silver nanoclusters based system (ChONC12-AgNCs) that is able to anchor at the Langmuir monolayer interface, which is mimicking the surface of living cells membrane.

Conformational rearrangements of G-quadruplex topology promoted by Cu(II) 12-MCCu(II)PyrAcHA-4 metallacrown.

Cu(II) 12-MCCu(II)PyrAcHA-4 metallacrown was studied by several spectroscopic techniques as an interacting ligand with G-quadruplex DNA structures. Investigations were performed on oligonucleotides bearing human telomeric and protooncogenic c-myc sequences in buffered solution mimicking ionic conditions in cellular environment. The planar square-based Cu(II) 12-MC-4 metallacrown interacts with GQ via an end-stacking mode with 1:1 stoichiometry. Circular dichroism (CD) titration revealed capability of this metallacrown to induce transformation of the GQ hybrid topology into the parallel form. Thermal melting experiment indicated higher thermal stability of both antiparallel (ΔTm = +15 °C) and parallel (ΔTm = ≥27 °C) G-quadruplexes in the presence of Cu (II) 12-MC-4. Indirect GQ FID assay let to determine high binding affinity of the Cu(II) 12-MC-4 to antiparallel 22Htel/Na+ GQ (KMC = 3.9 (±0.4) x 106 M-1). Comparing with lower binding constants previously reported for Ln (III) 15-MC-5 and Sm (III) 12-MC-4, one can conclude that the square planar geometry and the positive charge of metallacrown play an important role in MC/GQ interactions.

Monitoring of pH Using an i-Motif-Forming Sequence Containing a Fluorescent Cytosine Analogue, tC.

The i-motif is a four-stranded DNA structure formed from the cytosine (C)-rich ssDNA sequence, which is stabilized in slightly acidic pH. Additionally, labeling of a cytosine-rich sequence with a fluorescent molecule may constitute a way to construct a pH-sensitive biosensor. In this paper, we report tC-modified fluorescent probes that contain RET-related sequence C4GC4GC4GC4A. Results of the UV absorption melting experiments, circular dichroism (CD) spectra, and steady-state fluorescence measurements of tC-modified i-motifs are presented and discussed here. Efficient fluorescence quenching of tC fluorophore occurred upon lowering the pH from 8.0 to 5.5. Furthermore, we present and discuss fluorescence spectra of systems containing tC-modified i-motifs and complementary G-rich sequences in the ratios 1:1, 1:2, and 1:3 in response to pH changes. The fluorescence anisotropy was proposed for the study of conformational switching of the i-motif structure for tC-probes in the presence and absence of a complementary sequence. The possibility of using of the sensor for monitoring pH changes was demonstrated.

Practical Microwave Synthesis of Carbazole Aldehydes for the Development of DNA-Binding Ligands.

Microwave formylation of carbazole derivatives was investigated and 3-monoaldehydes were obtained in high yield. A potential DNA-binding ligand, 3-[(3-ethyl)-2-vinylbenzothiazolium]-9-N-ethyl carbazole iodide, was synthesized and characterized including spectral properties (UV-Vis absorption and fluorescence spectra). The binding selectivity and affinity of three carbazole ligands for double-stranded and G-quadruplex DNA structures were studied using a competitive dialysis method in sodium- and potassium-containing buffer solutions.

Binding Study of the Fluorescent Carbazole Derivative with Human Telomeric G-Quadruplexes.

The carbazole ligand 3 was synthesized, characterized and its binding interactions with human telomeric (22HT) G-quadruplex DNA in Na⁺ and K⁺-containing buffer were investigated by ultraviolet-visible (UV-Vis) spectrophotometry, fluorescence, circular dichroism (CD) spectroscopy, and DNA melting. The results showed that the studied carbazole ligand interacted and stabilized the intramolecular G-quadruplexes formed by the telomeric sequence in the presence of sodium and potassium ions. In the UV-Vis titration experiments a two-step complex formation between ligand and G-quadruplex was observed. Very low fluorescence intensity of the carbazole derivative in Tris HCl buffer in the presence of the NaCl or KCl increased significantly after addition of the 22HT G4 DNA. Binding stoichiometry of the ligand/G-quadruplex was investigated with absorbance-based Job plots. Carbazole ligand binds 22HT with about 2:1 stoichiometry in the presence of sodium and potassium ions. The binding mode appeared to be end-stacking with comparable binding constants of ~105 M-1 as determined from UV-Vis and fluorescence titrations data. The carbazole ligand is able to induce formation of G4 structure of 22HT in the absence of salt, which was proved by CD spectroscopy and melting studies. The derivative of carbazole 3 shows significantly higher cytotoxicity against breast cancer cells then for non-tumorigenic breast epithelial cells. The cytotoxic activity of ligand seems to be not associated with telomerase inhibition.

Cholesterol-Bearing Fluorescent G-Quadruplex Potassium Probes for Anchoring at the Langmuir Monolayer and Cell Membrane.

The purpose of the present work was to design, synthesize and spectrally characterize cholesterol-anchored fluorescent oligonucleotide probes (Ch(F-TBA-T), Ch(py-TBA-py)), based on G-quadruplexes, which were able to incorporate into a lipid structure (Langmuir monolayer, living cell membrane). The probes, based on the thrombin-binding aptamer (TBA) sequence, were labeled with fluorescent dyes which enabled simultaneous monitoring of the formation of G-quadruplex structures and visualization of probe incorporation into the cellular membrane. The combinations of fluorophores used included fluorescence resonance energy transfer (FRET) and excimer emission approaches. The structural changes of the probes upon binding with K⁺ or Na⁺ ions were monitored with fluorescence techniques. These systems showed a very high binding preference for K⁺ over Na⁺ ions. The use of confocal fluorescence microscopy indicated successful anchoring of the cholesterol-bearing fluorescent probes to the living cell membrane. These structurally simple cholesterol-based fluorescent probes have good potential for opening up new and exciting opportunities in the field of biosensors; e.g., in vivo detection of K⁺ ions.

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.

Carbazole Derivatives' Binding to c-KIT G-Quadruplex DNA.

The binding affinities of three carbazole derivatives to the intramolecular G-quadruplex (GQ) DNA formed by the sequence 5'-AGGGAGGGCGCTGGGAGGAGGG-3', derived from the c-KIT 1 oncogene region, were investigated. All carbazole cationic ligands that differed in the substituents on the nitrogen atom were able to stabilize G-quadruplex, as demonstrated using UV-Vis, fluorescence and CD spectroscopic techniques as well as molecular modeling. The spectrophotometric titration results showed spectral features characteristic of these ligands-bathochromic shifts and initial hypochromicity followed by hyperchromicity at higher GQ concentrations. All free carbazole ligands exhibited modest fluorescent properties, but after binding to the DNA the fluorescence intensity increased significantly. The binding affinities of carbazole ligands to the c-KIT 1 DNA were comparable showing values in the order of 105 M−1. Molecular modeling highlights the differences in interactions between each particular ligand and studied G-quadruplex, which potentially influenced binding strength. Obtained results relevant that all three investigated ligands have stabilization properties on studied G-quadruplex.

Carbazole ligands as c-myc G-quadruplex binders.

The interactions of c-myc G-quadruplex with three carbazole derivatives were investigated by UV-Vis spectrophotometry, fluorescence, CD spectroscopy, and molecular modeling. The results showed that a combination of carbazole scaffold functionalized with ethyl, triazole and imidazole groups resulted in stabilization of the intramolecular G-quadruplex formed by the DNA sequence derived from the NHE III1 region of c-myc oncogene (Pu22). Binding to the G-quadruplex Pu22 resulted in the significant increase in fluorescence intensity of complexed ligands 1-3. All ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex with comparable affinity, which agrees with binding model of end-stacking on terminal G-tetrads.

Sm(iii)[12-MCGa(III)shi-4] as a luminescent probe for G-quadruplex structures.

G-quadruplexes (GQs), spatial assemblies of guanine-rich DNA strands, play an important role in the regulation of gene expression and chromosome stabilization. These structures are recognized to be useful in cancer therapies as the presence of multiple G-quadruplexes in a telomeric strand stops cancer cell proliferation. Metallacrowns of the type 12-MC-4 form planar structures that have remarkable similarity to G-tetrads in terms of dimension, shape and the ability to bind alkali metal and lanthanide cations in a central cavity. The interaction between the Sm(iii)[12-MCGa(III)shi-4] (SmMC) metallacrown (MC) and human telomeric G-quadruplex structures was examined using several methods including CD titrations, CD melting temperatures, fluorescence titration of SmMC with GQ/Na+, fluorescence intercalator displacement (FID) assays and methods measuring the MC quenching effect on the Tb3+/GQ luminescence. It was proven that the studied metallacrown acted as a sensing probe and interacted with quadruplex DNA. The Stern-Volmer quenching constant (Kas) of Tb3+/GQ luminescence was calculated to be 3.9 × 105 M-1. The binding constant using the indirect FID method gave the result of 1.3 × 105 M-1. CD melting temperature experiments reveal the following pattern - the higher the concentration of the complex the lower the registered Tm for quadruplex DNA, which indicates a destabilizing effect of SmMC at higher GQ : MC ratios. These data implicate a shape and size selective interaction between MCs and GQs that may be exploited for telomere detection.

Studying the influence of stem composition in pH-sensitive molecular beacons onto their sensing properties.

Intracellular sensing using fluorescent molecular beacons is a potentially useful strategy for real-time, in vivo monitoring of important cellular events. This work is focused on evaluation of pyrene excimer signaling molecular beacons (MBs) for the monitoring of pH changes in vitro as well as inside living cells. The recognition element in our MB called pHSO (pH-sensitive oligonucleotide) is the loop enclosing cytosine-rich fragment that is able to form i-motif structure in a specific pH range. However, alteration of a sequence of the 6 base pairs containing stem of MB allowed the design of pHSO probes that exhibited different dynamic pH range and possessed slightly different transition midpoint between i-motif and open loop configuration. Moreover, this conformational transition was accompanied by spectral changes showing developed probes different pyrene excimer-monomer emission ratio triggered by pH changes. The potential of these MBs for intracellular pH sensing is demonstrated on the example of HeLa cells line.

Spectroscopic study of fluorescent probes based on G-quadruplex oligonucleotides labeled with ethynylpyrenyldeoxyuridine.

The design, synthesis, and spectral properties of four pyrene labeled oligonucleotide probes with G-quadruplex structure (Tel22-Tpy, Tel22-Upy, Tel22-6Upy, Tel22-18Upy) based on the 22-mer human telomeric sequence (Tel22) have been reported. Pyrene labels in the form of ethynylpyrenyldeoxyuridine have been inserted efficiently into oligodeoxynucleotides probes using phosphoramidite chemistry. The probes exhibited abilities to fold into G-quadruplex structures and to bind metal cations (Na+ and K+). Folding properties of probes and their spectral behavior were examined by recording the UV-vis, fluorescence, and CD spectra as well as by analyzing melting profiles. Fluorescence characteristics and G-quadruplex folding of probes were also studied at the interface of cationic dioctadecyldimethylammonium bromide (DODAB) monolayer. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter.

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.

Interaction of metallacrown complexes with G-quadruplex DNA.

Interactions of the G-quadruplex (GQ) DNA with two pentacoordinate lanthanide (III) metallacrown (MC) complexes containing phenylalanine hydroxamic acid (pheHA) and copper(II) ions of the formula Eu 15-[MCCu,pheHA]-5 (1) and Tb 15-[MCCu,pheHA]-5 (2) were investigated. Binding of both metallacrowns to human telomeric G-quadruplex DNA was followed using CD spectroscopy, DNA melting profiles, and fluorescent intercalator displacement (FID) assay. A new G-quadruplex binding assay based of quenching of Tb(III)-GQ luminescence was proposed and evaluated. All performed tests confirmed interactions of MCs with studied GQ structure. Binding affinities of MCs were appreciable (KMC ~2-5×10(5)M(-1)). Higher concentration of MCs (the ratio of GQ:MC above 2.5) caused destabilization of tetraplex structure of GQ as evidenced by CD spectroscopy, melting temperatures, and Tb(III)-GQ luminescence quenching results.

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.

Fluorescent Sensor for PH Monitoring Based on an i-Motif---Switching Aptamer Containing a Tricyclic Cytosine Analogue (tC).

There are cytosine-rich regions in the genome that bind protons with high specificity. Thus protonated C-rich sequence may undergo folding to tetraplex structures called i-motifs. Therefore, one can regard such specific C-rich oligonucleotides as aptamers that recognize protons and undergo conformational transitions. Proper labeling of the aptamer with a fluorescent tag constitutes a platform to construct a pH-sensitive aptasensor. Since the hemiprotonated C-C⁺ base pairs are responsible for the folded tetraplex structure of i-motif, we decided to substitute one of cytosines in an aptamer sequence with its fluorescent analogue, 1,3-diaza-2-oxophenothiazine (tC). In this paper we report on three tC-modified fluorescent probes that contain RET related sequences as a proton recognizing aptamer. Results of the circular dichroism (CD), UV absorption melting experiments, and steady-state fluorescence measurements of these tC-modified i-motif probes are presented and discussed. The pH-induced i-motif formation by the probes resulted in fluorescence quenching of tC fluorophore. Efficiency of quenching was related to the pH variations. Suitability of the sensor for monitoring pH changes was also demonstrated.

Pyrene functionalized molecular beacon with pH-sensitive i-motif in a loop.

In this work, we present a spectral characterization of pH-sensitive system, which combines the i-motif properties with the spatially sensitive fluorescence signal of pyrene molecules attached to hairpin ends. The excimer production (fluorescence max. ∼480 nm) by pyrene labels at the ends of the molecular beacon is driven by pH-dependent i-motif formation in the loop. To illustrate the performance and reversible work of our systems, we performed the experiments with repeatedly pH cycling between pH values of 7.5±0.3 and 6.5±0.3. The sensor gives analytical response in excimer-monomer switching mode in narrow pH range (1.5 pH units) and exhibits high pH resolution (0.1 pH unit).