Bernard Juskowiak

Nucleic acid-based fluorescent probes and their analytical potential

AbstractIt is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays. FigureHybridization with a nucleic acid target or affinity interactions with a nonnucleic acid target generate changes in the fluorescence characteristics of a nucleic acid-based fluorescent probe

Spectroscopic Characterization of i-motif Forming c-myc Derived Sequences Double-Labeled with Pyrene

In current studies we use the oligonucleotides based on c-myc sequence: CCC CAC CCT CCC CAC CCT CCC C (cmyc22) and CCC CAC CCT CCC CAC CCT CCC CA (cmyc22A) functionalized by pyrene moieties at both termini. Results of the circular dichroism (CD), UV absorption melting experiments, and steady-state fluorescence measurements of pyrene-modified i-motifs as well as their unlabeled precursors are presented and discussed here. The pyrene labels have a remarkable influence on i-motif stability which was deduced from CD spectra and confirmed by UV melting experiments. Both probes emit fluorescence band of pyrene monomer with intensity decreasing upon pH lowering.

Hemin/G-quadruplex structure and activity alteration induced by magnesium cations.

The influence of metal cations on G-quadruplex structure and peroxidase-mimicking DNAzyme activity was investigated. Experiments revealed a significant role of magnesium ion, which in the presence of potassium cation influenced DNAzyme activity. This ability has been associated with alteration of G-quadruplex topology and consequently affinity to bind hemin molecule. It has been demonstrated that G-quadruplex based on PS2.M sequence under these conditions formed parallel topology, which exhibited lower activity than that observed in standard potassium-containing solution. On the other hand DNAzyme/magnesium ion system based on telomeric sequence, which did not undergo significant structural changes, exhibited higher peroxidase activity upon magnesium ion addition. In both cases, the stabilization effect of magnesium cations on G-quadruplex structure was observed. The mechanism of DNAzyme activity alteration by magnesium ion can be explained by its influence on the pKa value of DNAzyme. Magnesium ion decreased pKa for PS2.M based system but increased it for telomeric DNAzyme. Magnesium cation effect on G-quadruplex structure as well as DNAzyme activity is particularly important since this ion is one of the most common metal cations in biological samples.

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).

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.

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.

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.

The interaction between G-quadruplex-forming oligonucleotide and cationic surfactant monolayer at the air/water interface.

We report on the interactions between a 21-mer quadruplex-forming oligonucleotide bearing human telomere sequence of dG(3)(T(2)AG(3))(3) (G4 DNA) and a positively charged dioctadecyldimethylammonium bromide (DODAB) monolayer at the air-aqueous interface, studied by surface film balance measurements. In the presence of G4 DNA, the π-A isotherm of the cationic Langmuir film shifted to lower molecular areas when compared with the reference isotherm recorded on the subphase containing only 50 mM triethylamine-acetate (TEAA) buffer. The presence of quadruplex-stabilizing metal cations (K(+) or Na(+)) further affected profiles of π-A isotherms. Further insight into processes related to the G4 DNA-monolayer interactions was provided by recording time profiles of the surface pressure of monolayer at a constant mean molecular area. In these experiments G4 DNA and/or metal ions were sequentially injected under the monolayer surface. Results indicated that multistranded assemblies of G4 DNA were formed at the monolayer interface even in the absence of metal ions, which suggested that the charged cationic surface of Langmuir monolayer induced aggregation of guanine-rich DNA strands. The presence of sodium and potassium ions inhibited formation of multi-stranded assemblies through the competitive G-quadruplex formation but to different extent that might be related to the differences in stability and topology of both quadruplexes.

Photoisomerizable arylstilbazolium ligands recognize parallel and antiparallel structures of G-quadruplexes.

The photoisomerization and DNA interaction studies of three arylstilbazolium derivatives with various samples of nucleic acids (duplexes, triplexes and tetraplexes) are reported. The equilibrium dialysis study revealed high binding affinities of ligands to tetraplex structures. The quadruplex-binding affinity could be switched by light, e.g., the E,E and E,Z isomers of 1,4-bis(vinylquinolinium)benzene (1) interacted with parallel and antiparallel tetraplexes exhibiting different binding selectivity. The E,Z-1 showed higher binding preference for c-myc DNA (a propeller-type quadruplex), whereas the E,E-1 favorably interacted with telomeric DNA (a basket-type quadruplex). The presence of quadruplex DNA hampered photoisomerization of quadruplex-bound ligand.

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.