Vibrational stark effect probes for nucleic acids

The vibrational Stark effect (VSE) has proven to be an effective method for the study of electric fields in proteins via the use of infrared probes. To explore the use of VSE in nucleic acids, we investigated the Stark spectroscopy of nine structurally diverse nucleosides. These nucleosides contained nitrile or azide probes in positions that correspond to both the major and minor grooves of DNA. The nitrile probes showed better characteristics and exhibited absorption frequencies over a broad range; that is, from 2253 cm-1 for 2'-O-cyanoethyl ribonucleosides 8 and 9 to 2102 cm(-1) for a 13C-labeled 5-thiocyanatomethyl-2'-deoxyuridine 3c. The largest Stark tuning rate observed was |Deltamu| = 1.1 cm(-1)/(MV/cm) for both 5-cyano-2'-deoxyuridine 1 and N2-nitrile-2'-deoxyguanosine 7. The latter is a particularly attractive probe because of its high extinction coefficient (epsilon = 412 M-1cm-1) and ease of incorporation into oligomers.     

生物应用锌(Ⅱ)荧光探针研究进展

本文对近年来生物应用锌(Ⅱ)荧光探针研究进展进行了较详细地评述,并对其化学规律、性质和某些局限性进行了讨论。     

Concentrated diffusing colloidal probes of Ca2+-dependent cadherin interactions

We report video microscopy measurements and inverse simulation analyses of specific Ca(2+)-dependent interactions between N-cadherin fragments attached to supported lipid bilayer-coated silica colloids in quasi-2D concentrated configurations. Our results include characterization of the bilayer formation and fluidity and the attachment of active extracellular cadherin fragments on bilayers. Direct measurements of interaction potentials show nonspecific macromolecular repulsion between cadherin fragments in the absence of Ca(2+) and irreversible bilayer fusion via cadherin-mediated attraction at >100 μM Ca(2+). Analysis of Ca(2+)-dependent N-cadherin bond formation in quasi-2D concentrated configurations using inverse Monte Carlo and Brownian Dynamics simulations show measurable attraction starting at 0.1 μM Ca(2+), a concentration significantly below previously reported values.     

Metal binding to ligand-containing peptide nucleic acids

The substitution of nucleobases in nucleic acid duplexes with ligands that have high affinity for transition metal ions creates metal-binding sites at specific locations within the duplexes. Several studies on the incorporation of metal ions into DNA and peptide nucleic acid (PNA) duplexes have suggested that the stability constant of the metal complex formed within the duplexes is a primary determinant of the thermal stability of the duplexes. To understand this relationship, we have synthesized two PNA monomers that carry the same ligand, namely 8-hydroxyquinoline, but have this ligand attached differently to the PNA backbone. The PNA monomers have been incorporated into PNA duplexes. UV and CD spectroscopy and calorimetric studies of the 8-hydroxyquinoline-PNA duplexes showed that the effect of the stability of the metal complex on the PNA duplexes was significantly modulated by the steric relationship between the complex and the duplex. This information is useful for the construction of hybrid inorganic-nucleic acid nanostructures.     

Charge transfer through modified peptide nucleic acids

We studied the charge transfer properties of bipyridine-modified peptide nucleic acid (PNA) in the absence and presence of Zn(II). Characterization of the PNA in solution showed that Zn(II) interacts with the bipyridine ligands, but the stability of the duplexes was not affected significantly by the binding of Zn(II). The charge transfer properties of these molecules were examined by electrochemistry for self-assembled monolayers of ferrocene-terminated PNAs and by conductive probe atomic force microscopy for cysteine-terminated PNAs. Both electrochemical and single molecular studies showed that the bipyridine modification and Zn(II) binding do not affect significantly the charge transfer of the PNA duplexes.     

Fluoroaromatic universal bases in peptide nucleic acids

Fluoroaromatic peptide nucleic acid residues were found to possess little base discrimination when incorporated into PNA.DNA double helices.     

Nitroazole universal bases in peptide nucleic acids

[formula: see text] The syntheses of PNA oligomers containing potential ambiguous nucleobase analogues, namely 3-nitropyrrole and 5-nitroindole, have been accomplished. Hybridization properties of these PNAs with complementary oligodeoxynucleotides were evaluated by thermal denaturation experiments. Both novel residues exhibited little variation in Tm (< or = 1.5 degrees C) when positioned against any of the four nucleoside bases. The capability to incorporate degenerate sites should further expand the utility of PNA in applications where precise sequence information is not available.     

Subcellular localised small molecule fluorescent probes to image mobile Zn2+

Zn²⁺, as the second most abundant d-block metal in the human body, plays an important role in a wide range of biological processes, and the dysfunction of its homeostasis is related to many diseases, including Type 2 diabetes, Alzheimer''s disease and prostate and breast cancers. Small molecule fluorescent probes, as effective tools for real-time imaging, have been widely used to study Zn²⁺ related processes. However, the failure to control their localisation in cells has limited their utility somewhat, as they are generally incapable of studying individual processes in a specific cellular location. This perspective presents an overview of the recent developments in specific organelle localised small molecule fluorescent Zn²⁺ probes and their application in biological milieu, which could help to extend our understanding of the mechanisms that cells use to respond to dysfunction of zinc homeostasis and its roles in disease initiation and development.

A number of recently developed subcellular localised small molecule fluorescent probes to image mobile Zn²⁺ are reviewed in this perspective.     

Sensitive determination of nucleic acids using organic nanoparticle fluorescence probes

This paper describes the preparation of organic nanoparticles by reprecipitation method under sonication and vigorous stirring. Transmission electron microscopy (TEM) was used to characterize the size and size distribution of the luminescent nanoparticles. Their average diameter was about 25 nm with a size variation of +/-18%. The fluorescence decay lifetime of the nanoparticles also was determined on a self-equipped fluorospectrometer with laser light source. The lifetime (approximately 0.09 micros) of nanoparticles is about three times long as that of the monomer. The nanoparticles were in abundant of hydrophilic groups, which increased their miscibility in aqueous solution. These organic nanoparticles have high photochemical stability, excellent resistance to chemical degradation and photodegradation, and a good fluorescence quantum yield (25%). The fluorescence can be efficiently quenched by nucleic acids. Based on the fluorescence quenching of nanoparticles, a fluorescence quenching method was developed for determination of microamounts of nucleic acids by using the nanoparticles as a new fluorescent probe. Under optimal conditions, maximum fluorescence quenching is produced, with maximum excitation and emission wavelengths of 345 and 402 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.4-19.0 microgml(-1) for calf thymus DNA (ct-DNA) and 0.3-19.0 microgml(-1) for fish sperm DNA (fs-DNA). The corresponding detection limits are 0.25 microgml(-1) for ct-DNA and 0.17 microgml(-1) for fs-DNA. The relative standard deviation of six replicate measurements is 1.3-2.1%. The method is simple, rapid and sensitive with wide linear range. The recovery and relative standard deviation are very satisfactory.     

6-Substituted quinoline-based ratiometric two-photon fluorescent probes for biological Zn2+ detection

New ratiometric two-photon fluorescent probes are developed from 6-substituted quinolines for biological Zn(2+) detection. They show large red shifts and good ratiometric responses upon Zn(2+) binding. They also exhibit high ion selectivities and large two-photon absorption cross sections at nearly 720 nm. Because the new probes are cell-permeable, they can be used to detect intracellular zinc flux under two-photon excitation.     

Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.     

Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development

Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.     

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.     

Analysis and purification of peptide nucleic acids by denaturing PAGE

A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.     

Convergent synthesis of peptide nucleic acids by native chemical ligation

[reaction: see text] A convergent strategy for synthesizing long contiguous PNA by a native chemical ligation-like technique of PNA segment couplings is presented. This approach required the synthesis of a new PNA-monomer featuring a 1-amino-2-thiol group. It is shown that the additional mercaptomethyl group leaves the hybridization properties of PNA ligation products unaffected. Furthermore, rapid and efficient fluorescence labeling of the ligation products is demonstrated.     

Thiazole orange as fluorescent universal base in peptide nucleic acids

Thiazole orange is shown to possess characteristics of a universal base while maintaining duplex stability. Its fluorescence properties allowed distinction between matched and single mismatched hybridisation.     

Simple, rapid and label-free colorimetric assay for Zn2+ based on unmodified gold nanoparticles and specific Zn2+ binding peptide

A simple strategy based on unmodified AuNPs and specific Zn(2+) binding peptide is exploited here as a new label-free mechanism for the rapid colorimetric Zn(2+) assay.     

Responsive polymer-based multicolor fluorescent probes for temperature and Zn2+ ions in aqueous media

We report on the fabrication of fluorescent and multicolor probes for Zn²⁺ ions and temperature from a mixture of three types of fluorophore-labeled responsive block copolymers in aqueous media. Quinoline-based Zn²⁺-recognizing fluorescent monomer ZQMA, red-emitting rhodamine B-based monomer RhBEA, and blue-emitting coumarin derivative Coum-OH, were synthesized first. A ZQMA-labeled well-defined double hydrophilic block copolymer (DHBC), PEG-b-P(MEO₂MA-co-ZQMA), was synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MEO₂MA) and ZQMA by utilizing a PEG-based macroRAFT agent. Following similar procedures, PEG-b-P(St-co-RhBEA) amphiphilic diblock copolymer and PEG-b-P(MEO₂MA-co-Coum) DHBC were also synthesized, where P(St-co-RhBEA) was a RhBEA-labeled polystyrene (PS) block. At room temperature in aqueous solution, almost nonfluorescent PEG-b-P(MEO₂MA-co-ZQMA) can effectively bind Zn²⁺ ions, leading to prominent green fluorescence enhancement due to the coordination of ZQMA with Zn²⁺ ions. However, by mixing red-emitting PEG-b-P(St-co-RhBEA) and blue-emitting PEG-b-P(MEO₂MA-co-Coum) with PEG-b-P(MEO₂MA-co-ZQMA) at an appropriate ratio, three color transitions could be observed. In the absence of Zn²⁺ ions, a mixed pink fluorescent originating from Coum and RhBEA was observed; upon the addition of a certain amount of Zn²⁺ ions, the green fluorescence enhanced dramatically, leading to a white fluorescence readout. By further increasing the amount of Zn²⁺ ions, the green fluorescence further enhanced and overwhelmed the blue and red emissions, leading to a green-dominant mixed-fluorescence emission. In addition, upon increasing the temperature, the fluorescence of Coum decreased considerably due to the fluorescence-resonance energy transfer (FRET) between Coum and ZQMA moieties. In this way, a ratiometric fluorescent thermometer can be constructed.