Detection of DNA hybridization by a pyrene-labeled polyelectrolyte prepared by ATRP

A novel pyrene-labeled polyelectrolyte (Py-PDMAEMA⁺) has been prepared by atom transfer radical polymerization (ATRP) for detection of DNA hybridization. The electrostatic and hydrophobic interactions between Py-PDMAEMA⁺ and ssDNA (hairpin or linear DNA) kept them forming a complex probe which could be used as a turn-off fluorescent sensor. The fluorescence intensity of the probe decreased upon adding the complementary strand, since the pyrene moiety could be intercalated into the duplex and thus the fluorescence quenching by nucleotide bases was strengthened. The proposed intercalation mode was confirmed by the circular dichroism spectra and the fluorescence quenching study with iodide. This pyrene-labeled polyelectrolyte combined with ssDNA may establish a novel fluorescence sensing system for DNA hybridization.     

Photo-Cross-Linking between BrU and Pyrene Residues in an RNA/DNA Hybrid

In this study, we investigated the photoreaction of ^BrU in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of ^BrU changed dramatically from hydrogen abstraction to cross-linking by changing the conformation of the duplex from the B-form to the A-form. Among three A-form structures, the largest amount of cross-linked products was observed when ^BrU was incorporated into the RNA strand and the pyrene was conjugated to the 5′ end of the DNA. These results indicate that the contact manner of pyrene was different between A- and B-form duplexes. This is a rare example of the use of the reactivity of bromouracil to analyze the contact between a small molecule with a weak binding affinity and a nucleic acid.     

The Binding of Monoclonal and Polyclonal Anti-Z-DNA Antibodies to DNA of Various Species Origin

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichia coli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.     

Fluorescence detection of single-nucleotide polymorphism with single-strand triplex-forming DNA probes

Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson-Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics.     

Effects of Metal Ions on Conductivity and Structure of Single DNA Molecule in Different Environmental Conditions

We design a novel nano-gap electrode to measure the current of DNA molecule, by which the current–voltage characteristics of individual native DNA, Ag-DNA and Ni-DNA molecules are obtained, respectively. The results show that the voltage gap of Ag- and Ni-DNA is higher than that of native DNA, and the conductance is lower than native DNA in neutral environment. The structure transition from B- to Z-DNA is observed in the presence of high concentrations of nickel ions and Ag-DNA appears chaos state by STM image and U-V spectra characterization. But in alkaline environment, the conductance of Ni-DNA rises and the voltage gap decreases with the increasing of nickel ion concentration denotes that the conductive ability of Ni-DNA is higher than that of native DNA.     

Aggregate Formation of Oligonucleotides that Assist Molecular Imaging for Tracking of the Oxygen Status in Tumor Tissue

The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA–ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor‐targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia‐selective manner. The oxygen‐dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.     

Multichromic polymers based on pyrene clicked thienylpyrrole

In this study, the effect of pendant pyrene on the optical and electronic properties of poly(2,5‐dithienylpyrrole)s was studied. For this purpose a new pyrene coupled 2,5‐dithienylpyrrole derivative (SNS‐pyrene) was synthesized through click reaction. SNS‐pyrene was electrochemically polymerized and its electrochemical and optical properties were investigated by electrochemical and optical techniques. The polymer had a band gap of 3.36 eV and displayed light green to blue color variation upon oxidation in less than 2.48 s. Additionally, electrochemical copolymerization of SNS‐pyrene with 3,4‐ethylenedioxythiophene was achieved whilst a detailed investigation was performed on the effect of electrochemical polymerization conditions on the optoelectronic properties of the copolymers. Studies revealed that the copolymers exhibit multichromic reversible redox behavior with lower band gaps and shorter switching times than their parent polymer, P(SNS‐pyrene) © 2014 Society of Chemical Industry.     

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis,Hybridization Studies,Fluorescent Properties and i‐Motif Stability

The synthesis of two new phosphoramidite building blocks for the incorporation of 5‐(pyren‐1‐yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene‐modified nucleobase component were found to destabilize an i‐motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene‐modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene‐modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady‐state fluorescence emission studies of oligonucleotides containing pyrene‐modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene‐modified UNA monomers was observed after formation of i‐motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene‐modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.     

Pyrene-modified unlocked nucleic acids: synthesis, thermodynamic studies, and fluorescent properties

Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools.     

Comparison of Animal and Plant DNA-Adducts after Exposure to Benzo(a)pyrene

The occurrence of abnormal hypermodified nucleotides on the DNA upon xenobiotic exposure has long been considered as a characteristic of carcinogenesis and mutagenesis in animal cells. We have previously shown that DNA adducts could also be formed in plants exposed to xenobiotics in natural and controlled conditions. In this study we have compared the DNA adducts formed in different animal species and in different plants after benzo(a)pyrene (B(a)P) exposure.

The main DNA adduct in mice stomach and skin correspond to the 7,8-diol 9,10-epoxide B(a)P-guanine. In liver from rat, fish and Xenopus, this adduct is detected, but is not the major one. In plants analyzed, this adduct is never formed. Ten different adducts are detected in plants. This result indicates that the metabolic pathway leading to genotoxic metabolites is different with species. In conclusion, the result suggest that risk assessment for human and environment due to genotoxic compounds should be realized using multiple species assays.     

3,3'-diethylthiatricarbocyanine iodide: a highly sensitive chiroptical reporter of DNA helicity and sequence

Using UV-vis absorption and circular dichroism (CD) spectroscopies, we explored the binding interactions of 3,3'-diethylthiatricarbocyanine iodide (Cy7) with polynucleotides of different sequences and helicity. CD showed to be a very diagnostic tool giving different spectroscopic chiroptical signatures for all explored DNA sequences upon Cy7 binding. Cy7 was able to spectroscopically discriminate between the right handed B-DNA of poly(dG-dC)(2) and its left handed Z-DNA counterpart induced by spermine or Co(III)hexamine via nearly opposite induced circular dichroic signal.     

Enzymatic synthesis of fluorescent oligomers assembled on a DNA backbone

A number of research laboratories have investigated the properties of multichromophore molecules and their applications in materials science and in biotechnology. Previous approaches for preparing such molecules have involved traditional organic synthesis. Here we describe the one-step enzymatic synthesis of such a multichromophore species by using a DNA-polymerizing enzyme (terminal deoxynucleotidyl transferase (TdT)). We find that a nucleotide-like molecule with pyrene replacing the DNA base (dPTP) can be accepted as a substrate for this enzyme to produce discrete chromophores that have 3 or 4 pyrenes consecutively, depending on which anomer (alpha or beta) is used. Products were characterized by gel electrophoresis, mass spectrometry, and fluorescence. The reaction was found to change the fluorescence emission of the chromophore from a maximum at 375 nm (the monomer nucleotide) to 490 nm in the oligomeric product. This new green-white emission is consistent with the formation of a pyrene excimer between adjacent pyrene glycosides, which exhibit a large Stokes shift of 130 nm. The enzymatic synthesis of the pyrene excimer might have applications in homogeneous biological assays for DNA fragments, such as those that arise during apoptosis.     

DNA nanotechnology: a future perspective

In addition to its genetic function, DNA is one of the most distinct and smart self-assembling nanomaterials. DNA nanotechnology exploits the predictable self-assembly of DNA oligonucleotides to design and assemble innovative and highly discrete nanostructures. Highly ordered DNA motifs are capable of providing an ultra-fine framework for the next generation of nanofabrications. The majority of these applications are based upon the complementarity of DNA base pairing: adenine with thymine, and guanine with cytosine. DNA provides an intelligent route for the creation of nanoarchitectures with programmable and predictable patterns. DNA strands twist along one helix for a number of bases before switching to the other helix by passing through a crossover junction. The association of two crossovers keeps the helices parallel and holds them tightly together, allowing the assembly of bigger structures. Because of the DNA molecule''s unique and novel characteristics, it can easily be applied in a vast variety of multidisciplinary research areas like biomedicine, computer science, nano/optoelectronics, and bionanotechnology.     

Fluorescent hybridization probes for sensitive and selective DNA and RNA detection

We outline the different approaches taken by our group in the design of fluorescent hybridization sensors. Molecular beacons (MBs) and binary probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.     

Fluorophore-Functionalized and Top-Covered Resorcin[4]arene Cavitands

This review describes our recent developments in the field of resorcin[4]arene cavitands. We present the syntheses of cavitands bearing pyrene, anthracene, and BODIPY dyes as fluorophores on the cavitand walls. These systems have been used to examine the conformational switching process between the closed vase and the open kite forms that are characteristic for this class of cavitands. In a second research direction, we prepared top-covered resorcin[4]arene-based, switchable container molecules, and investigated their binding and switching properties.     

Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

Selective switching of enzymatic activity has been a longstanding goal in synthetic biology. Drastic changes in activity upon mutational manipulation of the oligomerization state of enzymes have frequently been reported in the literature, but scarcely exploited for switching. Using geranylgeranylglyceryl phosphate synthase as a model, we demonstrate that catalytic activity can be efficiently controlled by exogenous modulation of the association state. We introduced a lysine-to-cysteine mutation, leading to the breakdown of the active hexamer into dimers with impaired catalytic efficiency. Addition of bromoethylamine chemically rescued the enzyme by restoring hexamerization and activity. As an alternative method, we incorporated the photosensitive unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) into the hexamerization interface. This again led to inactive dimers, but the hexameric state and activity could be recovered by UV-light induced cleavage of ONBY. For both approaches, we obtained switching factors greater than 350-fold, which compares favorably with previously reported activity changes that were caused by site-directed mutagenesis.     

Characterization of n-hexadecylalkylamine-intercalated graphite oxides as sorbents

Adsorption properties of graphite oxides hydrophobized by n-hexadecylamine, (C16)_xGO, were investigated using pyrene molecules as a model of nonionic organic contaminants. A large quantity of pyrene (28.5 mg/g) was adsorbed from a water-ethanol mixture (1:2) containing 2 mM of pyrene when (C16)_0.6GO was used. The isotherm of pyrene adsorption was better described by Freundlich equation rather than Langmuir equation, which indicated a single adsorption mechanism was involved. The change in the amount of adsorbed pyrene as a function of amine content in GO was very similar to that which occurs upon introduction of pyrene into (C16)_xGO films from chloroform solution, as determined by X-ray measurements. This suggests that pyrene molecules were adsorbed not only on the outer surface but also within the interlayer space of the intercalation compound. Swelling of the intercalation compound by ethanol can render the interlayers more organophilic and make access to hexadecylamine molecules bonded to the graphite oxide layer easier for pyrene molecules, especially in (C16)_xGOs with lower amine contents.     

2‐(1‐Ethynylpyrene)‐Adenosine as a Folding Probe for RNA—Pyrene in or out

A series of short RNA duplexes containing one or two 1‐ethynylpyrene‐modified adenine bases was synthesised. The melting behaviour of these duplexes was examined by monitoring temperature‐dependent pyrene fluorescence. In the singly modified RNA duplexes, the bases flanking the ethynylpyrene‐rA were varied to examine the sequence specificity of the fluorescence change of pyrene upon RNA hybridisation. Because an increase in pyrene fluorescence upon melting of the duplex can be correlated with intercalation of pyrene, and a decrease is usually associated with the position of pyrene outside the strand, a relationship between the flanking bases and the tendency of the dye to intercalate has been established. It was found that pyrene intercalation is less likely to take place if the modified base is flanked only by A–U base pairs. Flanking G–C base pairs, even only in the 5′‐direction of the modified base, will favour intercalation. In addition, we examined a doubly modified compound that had a pyrene located on each strand. The spectra indicated that the two pyrenes were close enough for interaction. Upon melting of the strand, a fluorescence blue shift corresponding to the dissociation of the pyrene–pyrene complex could be observed in addition to the intensity effect already known from the singly modified compounds. Two melting curves based on the different properties of the fluorophore could be extracted, leading to different melting points corresponding to the global duplex melting and to the change of local pyrene environment, respectively.     

Special Issue: A,B and Z: The Structure,Function and Genetics of Z-DNA and Z-RNA

It is now difficult to believe that a biological function for the left-handed Z-DNA and Z-RNA conformations was once controversial. The papers in this Special Issue, “Z-DNA and Z-RNA: from Physical Structure to Biological Function”, are based on presentations at the ABZ2021 meeting that was held virtually on 19 May 2021 and provide evidence for several biological functions of these structures. The first of its kind, this international conference gathered over 200 scientists from many disciplines to specifically address progress in research involving Z-DNA and Z-RNA. These high-energy left-handed conformers of B-DNA and A-RNA are associated with biological functions and disease outcomes, as evidenced from both mouse and human genetic studies. These alternative structures, referred to as “flipons”, form under physiological conditions, regulate type I interferon responses and induce necroptosis during viral infection. They can also stimulate genetic instability, resulting in adaptive evolution and diseases such as cancer. The meeting featured cutting-edge science that was, for the most part, unpublished. We plan for the ABZ meeting to reconvene in 2022.     

BENZO(A)PYRENE METABOLISM: ROLE OF BIOALKYLATION

Previous studies of the metabolic activation of chemical carcinogens including benzo(a)pyrene have demonstrated that selected aromatic hydrocarbons can undergo a series of biological substitution reactions in tissue cytosol preparations when in the presence of the methyl donor S-adenosyl-L-methionine. Although identification of the methylated metabolites of carcinogens had been tentatively established using liquid chromatographic techniques, confirmation of the identity of these methylated derivatives has remained elusive. In the present study, the carcinogen benzo(a)pyrene was found to undergo methylation reactions in the presence of rat liver cytosol preparations to yield products that were identified as methylated, formyl and hydroxyalkyl derivatives of the parent carcinogen. Identification and confirmation of the identity of the 6-methylbenzo(a)pyrene metabolite was confirmed by both UV detection, gas chromatographic and mass spectral identification, and NMR analysis of metabolic fractions obtained from incubations of benzo(a)pyrene. The results conclusively demonstrate that the carcinogen benzo(a)pyrene undergoes biological substitution reactions to yield 6-methylbenzo(a)pyrene in the presence of cytosolic enzymes. The presence of 6-methylbenzo(a)pyrene as a metabolite of benzo(a)pyrene illustrates and confirms the presence of centers of reactivity in unsubstituted aromatic hydrocarbons. These centers of reactivity serve as sites for biochemical substitution reactions, yielding methyl substituted hydrocarbons. Furthermore, these methylated metabolites of polycyclic aromatic hydrocarbons are further metabolically activated to hydroxyalkyl and formyl derivatives which can be subsequently activated to electrophilic esters leading to DNA and protein reactive species.