DNA-decorated nanoparticles as nanosensors for rapid detection of ascorbic acid

We designed an assay for rapid detection of ascorbic acid (AA) with a DNAzyme cleaving its DNA substrate in the presence of Cu(2+) and AA. The sensor consists of two DNA strands that form a complex between each other. The 5'-end of the DNAzyme binds the substrate DNA via Watson-Crick bonding and the 3'-end binds through formation of a DNA-triplex via Hoogsteen hydrogen bonding. The substrate DNA was prepared by two different methods. In the first case the nucleic acid was modified with fluorescein/dabcyl FRET pair across the cleavage site. In the second case the nucleic acid modified with fluorescein was immobilised on gold nanoparticles. DNAzyme contains a loop forming a complex with Cu(2+) ions. The oxidation of ascorbic acid (AA) with oxygen yields hydrogen peroxide. The latter interacts with Cu(2+) to give hydroxyl radicals. They break substrate DNA in close vicinity to the copper/DNA complex to separate fluorescein from gold nanoparticles leading to the increase in fluorescence intensity. Use of substrate DNA modified with the fluorescein/dabcyl couple allowed to measure AA concentration within 3 min with the detection limit of 2.5 μM. Employment of gold nanoparticles decorated with fluorescein-modified DNA allowed to improve the detection limit of AA quantification by two orders of magnitude due to enhanced cleavage of DNA catalysed by Au clusters. Fructose, sucrose, glucose, urea, and citric acid did not interfere with our assay even at concentration of 1mM. Good selectivity allowed us to apply our rapid and sensitive assays to detection of AA in vitamin C tablets, urine and orange juice.     

Optimization of acetonitrile co-solvent and copper stoichiometry for pseudo-ligandless click chemistry with nucleic acids

The copper(I) catalyzed azide-alkyne cycloaddition 'click' reaction yields a specific product under mild conditions and in some of the most chemically complex environments. This reaction has been used extensively to tag DNA, proteins, glycans and only recently RNA. Click reactions in aqueous buffer typically include a ligand for Cu(I), however we find that acetonitrile as a minor co-solvent can serve this role. Here we investigate the click labeling of RNA and DNA in aqueous buffer to determine the relationship between the stoichoimetry of Cu(I) and the acetonitrile co-solvent that affects nucleic acid stability. We find that very low concentrations of acetonitrile perform equally well and obviate the need for any additional Cu(I) stabilizing ligand. These pseudo-ligandless reaction conditions are optimal for nucleic acids click conjugations.     

Degradation of Single Stranded Nucleic Acids by the Chemical Nuclease Activity of the Metal Complex [Cu(phen)(nal)]+

The chemical design of metal complexes of the type [Cu(phen)(antib)]⁺ (where antib is a quinolone or a fluoroquinolone) has been carried out in an approach to better understand how the coordination of their components affect the activity of quinolones. The ability of [Cu(phen)(nal)]⁺ to interact with DNA in vivo and its capacity to promote the degradation of plasmid and chromosomal DNA, under reductive conditions has been previously reported. However whether this compound utilizes other intracellular targets to promote bacterial killing was a question that deserved to be answered. In this paper, the studies of the chemical nuclease properties encoded by the metal complex [Cu(phen)(nal)]⁺ were extended by using different types of single chain nucleic acids, i.e, ribosomal and tumor mosaic virus RNAs as well as poly-dA-dT. Our results showed that degradation of the nucleic acids occurred only under reductive conditions. Although MPA and [3-mercaptoethanol were the chemical reducers that best assisted the nuclease reaction, other biological compounds such as citric and succinic acid also were shown to act like reducers in that reaction. All.hough the nuclease activity of [Cu(phen)(nal)]⁺ was comparable to that exhibited by bis copper phenanthroline [Cu(phen)z]²⁺our results showed that none of the individual components of [Cu(phen)(nal)]⁺ was able to promote the degradation of either the RNAs or poly(dA-dT). These results strongly support the hypothesis that the metal complex [Cu(phen)(nal)] uses not only DNA but also RNA as targets to promote bacterial killing.     

Two-dimensional strandness-dependent electrophoresis: a method to characterize single-stranded DNA, double-stranded DNA, and RNA-DNA hybrids in complex samples

We describe two-dimensional strandness-dependent electrophoresis (2D-SDE) for quantification and length distribution analysis of single-stranded (ss) DNA fragments, double-stranded (ds) DNA fragments, RNA-DNA hybrids, and nicked DNA fragments in complex samples. In the first dimension nucleic acid molecules are separated based on strandness and length in the presence of 7 M urea. After the first-dimension electrophoresis all nucleic acid fragments are heat denatured in the gel. During the second-dimension electrophoresis all nucleic acid fragments are single-stranded and migrate according to length. 2D-SDE takes about 90 min and requires only basic skills and equipment. We show that 2D-SDE has many applications in analyzing complex nucleic acid samples including (1) estimation of renaturation efficiency and kinetics, (2) monitoring cDNA synthesis, (3) detection of nicked DNA fragments, and (4) estimation of quality and in vitro damage of nucleic acid samples. Results from 2D-SDE should be useful to validate techniques such as complex polymerase chain reaction, subtractive hybridization, cDNA synthesis, cDNA normalization, and microarray analysis. 2D-SDE could also be used, e.g., to characterize biological nucleic acid samples. Information obtained with 2D-SDE cannot be readily obtained with other methods. 2D-SDE can be used for preparative isolation of ssDNA fragments, dsDNA fragments, and RNA-DNA hybrids.     

Kinetics and equilibria for the formation of a new DNA metal-intercalator: the cyclic polyamine Neotrien/copper(II) complex

A study has been performed of the kinetics and equilibria involved in complex formation between the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) and Cu(II) in acidic aqueous solution and ionic strength 0.5 M (NaCl), by means of the stopped-flow method and UV spectrophotometry. Spectrophotometric titrations and kinetic experiments revealed that the binding of Cu(II) to Neotrien gives rise to several 1:1 complexes differing in their degree of protonation. Under the experimental hydrogen ion concentration range investigated, complexation occurs by two parallel paths: (a) M2+ + (H4L)4+ <==> (MH4L)6+ and (b) M2+ + (H3L)3+ <==> (MH3L)5+. The rate constants values found for complex formation, by paths (a) and (b), are much lower than the values expected from water exchange at copper(II) and other amine/Cu(II) complexation kinetic constants. Kinetic experiments at different NaCl concentrations indicated that this finding was not due to chloride ion competition in complex formation with Neotrien, but it was related to a ring rigidity effect. As the phenanthroline moiety could, in principle, interact with nucleic acids by intercalation or external binding, some preliminary measurements concerned with the possible interactions occurring between the Cu(II)/Neotrien complex and calf thymus DNA (CT-DNA) have also been carried out. The absorption spectra of the Cu(II)/Neotrien complex change upon addition of CT-DNA at pH 7.0, revealing the occurrence of complex-nucleic acid interactions. Moreover, fluorescence titrations, carried out by adding the Cu(II)/Neotrien complex to CT-DNA, previously saturated with ethidium bromide (EB), show that the Cu(II)/Neotrien complex is able to displace EB from DNA, suggesting the complex is able to intercalate into the polynucleotide and then to cleave the phosphodiester bond of DNA.     

NAMOT2--a redesigned nucleic acid modeling tool: construction of non-canonical DNA structures

Using a new set of reduced coordinates developed for describingregular and unusual nucleic acid structures, we have revisedour nucleic acid modeling tool NAMOT2. NAMOT2 is general interms of modeling different nucleic acid structures. A set ofmodifiable libraries allows users to customize their modelingenvironment. With this set of libraries, NAMOT2 can be usedto model non-canonical structures such as parallel-stranded,triple-stranded and quadruple-stranded nucleic acid molecules.For modeling irregular structures (junctions, hairpin loops,etc.), we introduce a structural recipe approach. The completeprocedure using NAMOT2 to construct the structure of a specificmolecule is treated as the recipe for that structural motif.The existing recipes can be modified to generate new recipesfor different structural motifs. Several examples of nucleicacids with non-canonical structures were modeled using NAMOT2.These examples include a DNA-drug complex, a DNA cube, a six-armjunction and a curved DNA molecule.     

Immunochemical studies of the O-antigens of Vibrio cholerae. The constitution of a lipopolysaccharide from V. cholerae 569B (Inaba)

Binding of Cu(2+) to polynucleotides has been found to occur at 1 Cu/5 polymer P and at similar molar ratios of copper to selected nucleic acid derivatives. Electron spin resonance (ESR) measurements show that the g-values and the hyperfine splitting constants vary substantially among the different complexes. Binding of Cu(2+) to the phosphates as well as to the bases is observed, and in some cases occurs simultaneously.     

An ultrasensitive photoelectrochemical nucleic acid biosensor

A simple and ultrasensitive procedure for non-labeling detection of nucleic acids is described in this study. It is based on the photoelectrochemical detection of target nucleic acids by forming a nucleic acid/photoreporter adduct layer on an ITO electrode. The target nucleic acids were hybridized with immobilized oligonucleotide capture probes on the ITO electrode. A subsequent binding of a photoreporter--a photoactive threading bis-intercalator consisting of two N,N'-bis(3-propyl-imidazole)-1,4,5,8-naphthalene diimides (PIND) linked by a Ru (bpy)2(2) (bpy = 2,2'-bipyridine) complex (PIND-Ru-PIND)-allowed for photoelectrochemical detection of the target nucleic acids. The extremely low dissociation rate of the adduct and the highly reversible photoelectrochemical response under visible light illumination (490 nm) make it possible to conduct nucleic acid detection, with a sensitivity enhancement of four orders of magnitude over voltammetry. These results demonstrate for the first time the potential of photoelectrochemical biosensors for PCR-free ultrasensitive detection of nucleic acids.     

Identification of nucleic acid binding sites on translin-associated factor X (TRAX) protein

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.     

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid–NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA–protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA–protein complex, reduce the nucleic acid chaperone activity of NdAg.     

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities, in poly(deoxythymidylic acid)-poly-L-lysine complexes

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time tau -R = 2 ns, and the other one by a tau -R greater than or equal to 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Interactions of water-soluble metalloporphyrins with nucleic acids studied by resonance Raman spectroscopy.

The resonance Raman spectra of water-soluble porphyrins, M(TMpy-P4) (M = Cu(II), Ni(II) and Co(III] and their mixtures with poly(dG-dC)2, poly(dA-dT)2 and calf thymus and salmon DNAs were measured using a divided rotating cell to determine the magnitudes of frequency shift and intensity variation resulting from M(TMpy-P4)-nucleic acid interactions. Bands II(C beta-H bending, approximately 1100 cm-1) and VIII(C beta-C beta stretch, approximately 1570 cm-1) show a large and small upward shift, respectively, when Cu(TMpy-P4) and Ni(TMpy-P4) are intercalated at the G-C sites. In contrast, these bands show a small upward and downward shift, respectively, when Co(TMpy-P4) is groove-bound at the A-T sites of nucleic acids. Both Bands V (approximately 1260 cm-1) and IX (approximately 1646 cm-1) which originate in the N-methylpyridyl group always show small downward shifts due to coulombic interaction between the N-CH3+ group of TMpy-P4 and the PO2 group of the nucleic acid.     

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.     

A high-throughput screening method for amino acid dehydrogenase

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)–calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)–calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations.     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids

The interaction between the broad-spectrum antimicrobial agent, polyhexamethylene biguanide (PHMB), and various nucleic acids was investigated. Titration of either single- or double-stranded 100-bp DNA, or mixed-molecular weight marker DNA, or tRNA with PHMB caused precipitation of a complex between nucleic acid and PHMB in which the nucleotide/biguanide ratio was always close to unity. Binding of PHMB was highly cooperative, with apparent Hill coefficients 10.3-14.6. When a fluorescent derivative of PHMB was titrated with increasing amounts of nucleic acid, all four forms of nucleic acid caused strong polarisation of fluorescence, demonstrating the association with PHMB. The intensity and broad-spectrum binding of PHMB to all forms of nucleic acid has significant implications for the mechanism of action of this biocide.     

A "turn-on" fluorescent copper biosensor based on DNA cleavage-dependent graphene-quenched DNAzyme

A novel and promising "turn-on" fluorescent Cu(2+) biosensor is designed based on graphene-DNAzyme catalytic beacon. Due to the essential surface and quenching properties of two-dimensional graphene, it can function as both "scaffold" and "quencher" of the Cu(2+)-dependent DNAzyme, facilitating the formation of self-assembled graphene-quenched DNAzyme complex. However, Cu(2+)-induced catalytic reaction disturbs the graphene-DNAzyme conformation, which will produce internal DNA cleavage-dependent effect. In this case, the quenched fluorescence in graphene-DNAzyme is quickly recovered to a large extent in 15 min. Compared with common DNAzyme-based sensors, the presented graphene-based catalytic beacon greatly improves the signal-to-background ratio, hence increasing the sensitivity (LOD=0.365 nM). Furthermore, the controllable DNA cleavage reaction provides an original and alternative internal method to regulate the interaction between graphene and DNA relative to the previous external sequence-specific hybridization-dependent regulation, which will open new opportunities for nucleic studies and sensing applications in the future.     

Toward protein-cleaving catalytic drugs: artificial protease selective for myoglobin

A protein-cleaving catalyst highly selective for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin (Mb) was designed by attaching the Cu(II) or Co(III) complex of cyclen to a binding site searched by a combinatorial method using peptide nucleic acid monomers as building units. Various linkers were inserted between the catalytic Co(III) center and the binding site of the Mb-cleaving catalyst. Kinetic data revealed catalytic turnover of the Mb cleavage by the Cu(II) or Co(III) complex. MALDI-TOF MS revealed cleavage of the polypeptide backbone of Mb at selected positions. N-Terminal sequencing of the cleavage products identified the cleavage site and provided evidence for the hydrolytic nature of the Mb cleavage. Various chelating ligands were tested as the ligand for the Co(III) center of the Mb-cleaving catalyst. Among the nine chelating ligands examined, only cyclen and its triaza-monooxo analogue manifested catalytic activity.