Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

Functional polymer/inorganic hybrid nanoparticles for macrophage targeting delivery of oligodeoxynucleotides in cancer immunotherapy

To efficiently deliver CpG oligodeoxynucleotides (ODN) in cancer immunotherapy, a multifunctional macrophage targeting delivery system was designed and prepared. Mannosylated carboxymethyl chitosan/protamine sulfate/CaCO₃/ODN (MCMC/PS/CaCO₃/ODN) nanoparticles were prepared using a facile self-assembly method. The functional components, including MCMC to endow the nanoparticles with macrophage targeting ability, PS to improve the ODN loading capacity and enhance the cell uptake, and CaCO₃ to encapsulate ODN and induce the favorable pH sensitivity, were introduced to the delivery systems by self-assembly. Due to the mannose mediated endocytosis and the favorable effects of PS in overcoming delivery barriers, MCMC/PS/CaCO₃/ODN nanoparticles exhibit a much higher ODN delivery efficiency and a significantly enhanced immune stimulation capacity as compared with Lipofectamine 2000/ODN complexes. The regulation of NF-κB activity by our ODN delivery system results in dramatically increased production of proinflammatory cytokines including IL-12, IL-6, and TNF-α in RAW264.7 cells. The significantly increased CD80 expression after stimulation by the ODN delivery systems indicates the successful modulation of the macrophage polarity to the anti-tumor M1 phenotype. The multifunctional macrophage targeting delivery system developed has promising applications in delivery of CpG ODN in cancer immunotherapy.     

Voltammetry of Osmium End‐Labeled Oligodeoxynucleotides at Carbon,Mercury, and Gold Electrodes

Voltammetric behavior of oligodeoxynucleotide (ODN) 5′‐T₄₀ (GAA)₇–3′ end‐labeled with osmium tetroxide,2,2‐bipyridine [Os(VIII)bipy] was compared with Os(VIII)bipy‐base‐ and with Os(VI)bipy‐sugar‐modified thymine ribosides. Cyclic voltammograms of Os(VIII)bipy‐modified ODN at mercury and carbon electrodes were similar but not identical to those of Os(VIII)bipy‐modified thymine riboside. Treatment of the ODN with Os(VI)bipy did not result in the ODN modification, in agreement with the known specificity of the reagent to the sugar cis‐diols. We show that in addition to mercury and carbon electrodes, the gold electrode can be used to detect Os(VIII)bipy‐labeled ODN. Comparison of voltammetric behavior of end‐labeled ODN using three types of electrodes most frequently used in DNA analysis may help to optimize electrochemical DNA sensors.     

Probing DNA Mismatched and Bulged Structures by Using 19F NMR Spectroscopy and Oligodeoxynucleotides with an 19F‐Labeled Nucleobase

In this study, DNA local structures with bulged bases and mismatched base pairs as well as ordinary full‐matched base pairs by using ¹⁹F NMR spectroscopy with ¹⁹F‐labeled oligodeoxynucleotides (ODNs) were monitored. The chemical shift change in the ¹⁹F NMR spectra allowed discrimination of the DNA structures. Two types of ODNs possessing the bis(trifluoromethyl)benzene unit (F‐unit) at specified uridines were prepared and hybridized with their complementary or noncomplementary strands to form matched, mismatched, or bulged duplexes. By using ODN F1, in which an F‐unit was connected directly to a propargyl amine‐substituted uridine, three local structures, that is, full‐matched, G–U mismatch, and A‐bulge could be analyzed, whereas other structures could not be discriminated. A molecular modeling study revealed that the F‐unit in ODN F1 interacted little with the nucleobases and sugar backbone of the opposite strand because the linker length between the F‐unit and the uridine base was too short. Therefore, the capacity of ODN F1 to discriminate the DNA local structures was limited. Thus, ODN F2 was designed to improve this system; aminobenzoic acid was inserted between the F‐unit and uridine base so the F‐unit could interact more closely with the opposite strand. Eventually, the G‐bulge and T–U mismatch and the three aforementioned local structures could be discriminated by using ODN F2. In addition, the dissociation processes of these duplexes could be monitored concurrently by ¹⁹F NMR spectroscopy.     

Template-directed photoreversible ligation of DNA via 7-carboxyvinyl-7-deaza-2′-deoxyadenosine

An efficient template-directed photoligation of oligodeoxynucleotide (ODN) using 7-deaza-2′-deoxyadenosine derivative ^VZA is described. When ODN containing ^VZA at the 5′ end was photoirradiated with ODNs containing a pyrimidine base at the 3′ end in the presence of template ODN, rapid and efficient ligation (cycloaddition reaction) was observed without any byproduct formation. ODNs containing ^VZA showed an extremely high reactivity as compared with those reported in previous photoligations.     

Specific hydrolysis of DNA by Ce<Superscript>4+</Superscript>-ODN hybrid

The cerium complex bound to the derivative of oligoDNA has been synthesized successfully that can hydrolyze DNA with sequence-specificity. The synthesized derivative of oligoDNA, 5′-EDTA-P-10 mers ODN, was demonstrated by HPLC. The fluorescence spectrum of Tb³⁺ was detected after its interaction with the hybrid of 10-mers ODN and 26-mers ODN and the results show that the artificial endo-enzyme can recognize and combine firmly with the substrate DNA. The electrophorogram shows that the cerium-oligoDNA hybrid can specifically hydrolyze its substrate DNA and the cleavage site of this hydrolysis reaction is also discussed. This artificial nuclease can be widely used in molecular biology and genetic engineering as one kind of endo-enzyme.     

Scanning Electrochemical Microscopy (SECM) Based Detection of Oligonucleotide Hybridization and Simultaneous Determination of the Surface Concentration of Immobilized Oligonucleotides on Gold

The direct mode of scanning electrochemical microscopy (SECM) was used for the local deposition of oligonucleotide (ODN) patterns on thin gold films and the generation‐collection (GC) mode was applied for the determining the amount of surface‐accessible oligonucleotides. The local deposition was achieved through the micrometer‐sized formation of a conducting polymer bearing 15^mer single‐stranded oligonucleotide strands. After the interaction of the oligonucleotide with its biotin‐labeled complimentary strand, streptavidin was bound. The molecular assembly was completed by linking biotin‐labeled β‐galactosidase from Escherichia coli to the streptavidin. The activity of the linked β‐galactosidase was mapped with SECM in the GC mode by monitoring the oxidation of p‐aminophenol (PAP) formed in the enzyme‐catalyzed hydrolysis of p‐aminophenyl‐β‐D ‐galactopyranoside. The feedback effect due to recycling of the reaction product at the gold surface was analyzed. It was shown experimentally that this effect becomes insignificant at ultramicroelectrode (UME)‐substrate distances larger than 3 UME radii. The flux of formed PAP allowed the determination the surface density of accessible oligonucleotide strands in the functionalized polymer. It was shown that that thicker pyrrole/ODN–Pyrrole polymer films do not lead to a significantly increased accessible ODN surface concentration.     

Fluorescence detection of cytosine/guanine transversion based on a hydrogen bond forming ligand

In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate potential use of a hydrogen bond forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), for the fluorescence detection of the cytosine (C)/guanine (G) mutation sequence of the cancer repression gene p53. Our method is based on construction of the AP site in ODN duplexes, which allows small synthetic ligands to bind to target nucleobases accompanied by fluorescence signaling: an AP site-containing ODN is hybridized with a target ODN so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleobases through hydrogen-bonding. In 10 mM sodium cacodylate buffer solutions (pH, 7.0) containing 100 mM NaCl and 1.0 mM EDTA, AMND is found to strongly bind to C (K_d=1.5×10⁻⁶ M) in the target ODN while the binding affinity for G is relatively moderate (K_d=50×10⁻⁶ M). Significant fluorescence quenching of AMND is observed only when binding to C, making it possible to judge the C/G transversion with the naked eye.     

Oligodeoxynucleotide-modified capillary for electrophoretic separation of single-stranded DNAs with a single-base difference.

We describe here a method of affinity capillary electrophoresis in which oligodeoxynucleotide (ODN) was immobilized onto the inner surface of the capillary. The immobilized ODN functioned successfully as an affinity ligand for sequence-based DNA separation. Six- or 12-mer ODN with a sequence complementary to one of the c-K-ras gene was used as an immobilized ligand. When the 12-mer ODN was used, the detection peak for the complementary ODN disappeared selectively, while the single-base mutant was detected as usual. In contrast, when the 6-mer ODN was used as the affinity ligand with a mixture of the complementary ODN and its single-base mutant, it was possible to detect both as completely separate peaks. That is, the separation mode was dependent on the base number of the immobilized ODN used as an affinity ligand.     

C4’-Fluorinated Oligodeoxynucleotides: Synthesis,Stability, Structural Studies

Fluoro-substitution on the ribose moiety (e. g., 2’-F-deoxyribonucleotide) represents a popular way to modulate the ribose conformation and, hence, the structure and function of nucleic acids. In the present study, we synthesized 4’-F-deoxythymidine (^4’-FT) and introduced it to oligodeoxyribonucleotides (ODNs). Though scission of the glycosylic bond of ^4’-FT followed by strand cleavage occurred to some extent under alkaline conditions, the ^4’-FT-modified ODNs were rather stable in neutral buffers. NMR studies showed that like 2’-F-deoxyribonucleoside, ^4’-FT exists predominantly in the North conformation not only in the nucleoside form but also in the context of ODN strands. Circular dichroism spectroscopy, thermal denaturing and RNase H1 footprinting studies of ^4’-FT-modified ODN/cDNA and ODN/cRNA duplexes indicated that the North conformation tendency of ^4’-FT is maintained in the duplexes, leading to a local structural perturbation. Collectively, 4’-F-deoxyribonucleotide structurally resembles the 2’-F-deoxyribonucleotide but imparts less structural perturbation to the duplex than the latter.     

Effect of base sequence and deprotonation of Guanine cation radical in DNA

The deprotonation of guanine cation radical (G+*) in oligonucleotides (ODNs) was measured spectroscopically by nanosecond pulse radiolysis. The G+* in ODN, produced by oxidation with SO4-*, deprotonates to form the neutral G radical (G(-H)*). In experiments using 5-substituted cytosine-modified ODN, substitution of the cytosine C5 hydrogen by a methyl group increased the rate constant of deprotonation, whereas replacement by bromine decreased the rate constant. Kinetic solvent isotope effects on the kinetics of deoxyguanosine (dG) and ODN duplexes were examined in H2O and D2O. The rate constant of formation of G(-H)* in dG was 1.7-fold larger in H2O than D2O, whereas the rate constant in the ODN duplex was 3.8-fold larger in H2O than D2O. These results suggest that the formation of G(-H)* from G+* in the ODN corresponds to the deprotonation of the oxidized hydrogen-bridged (G+*-C) base pair by a water molecule. The characteristic absorption maxima of G+* around 400 nm were shifted to a longer wavelength in the order of G相似文献   

Comparative Electrochemical and Spectroscopic Studies of I‐Motif‐forming DNA Nonamers

The paper shows the structural diversity of cytosine (C)‐rich oligodeoxynucleotides (ODNs) arising from their detail nucleotide sequence and experimental conditions. In slightly acidic solutions, the ODN nonamers with different adenine (A) and cytosine (C) sequences can adopt non‐canonical structures involving protonated bases. A distinct secondary structure formed in (C)‐rich sequences, called i‐motif (iM), consists of hemiprotonated and intercalated cytosine base pairs (C.C⁺). Folding and unfolding of particular structures in solutions were monitored by ¹H NMR and CD spectroscopies and native polyacrylamide gel electrophoresis (PAGE), which are capable to determine their structural characteristics. Effects of sequences and their proclivity to formation of the iM on electrochemical behaviour of the ODN nonamers were studied by electrochemical methods. The LSV signals of A and C obtained from the reductive dissolution of ODN adsorption layers on a hanging mercury drop electrode were processed by elimination voltammetry with linear scan (EVLS), which revealed complex effects of the nonamer properties (namely their primary and secondary structure confirmed in solution) on their adsorption and reduction activity.     

Investigation of surface oligonucleotide-binding proteins of eucaryotic cells by affinity modification with reactive oligonucleotide derivatives

Interactions between oligodeoxyribonucleotides (ODN) with different sequences and cell proteins were examined using the affinity modification by [³²P]-labeled reactive oligonucleotide derivatives. 3"-Terminal ribouridine oxidized with sodium periodate, 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine, and the maleimide residue were used as reactive groups. All the compounds used are specific reagents. The set of the discovered nucleic acid-binding (NA-binding) proteins depends on the chemical properties of the affinity reagent. The presence of the hydrophobic group at the 5"-terminus of the ODN molecule is the key factor determining the variety of the discovered NA-binding proteins. The cells of different origin (A431, HeLa, KB, MCF-7, Hap-2, K562, Cos-7, NIH/3T3, human-lung primary epithelial cells, and porcine kidney primary cells) are characterized by the same set of NA-binding proteins whose affinity modifications depends on the conditions of incubation of oligonucleotides with the cells. Treatments of cells disturbing the integrity of the cellular membrane (scrapping, treatment with trypsin, or cell permeabilization with streptolysin O or saponin), disrupt interactions between NA-binding proteins from native cells and ODN.     

Sequence- and base-specific delivery of nitric oxide to cytidine and 5-methylcytidine leading to efficient deamination

Nitric oxide (NO) is an important endogenous regulatory molecule, and S-nitrosothiols are believed to play a significant role in NO storage, transport, and delivery. On the basis of their ability to generate NO in vivo, S-nitrosothiols can be used as therapeutic drugs. In this study, we have developed an innovative method for sequence- and base-specific delivery of NO to a specific site of DNA followed by specific deamination. We designed a NO transfer reaction from S-nitroso thioguanine to an imino tautomer of cytosine. Nitrosation of the thioguanosine-containing ODN 1 was carried out with S-nitroso-N-acetylpenicillamine (SNAP) to produce ODN 2. An interstrand NO transfer reaction was performed using ODN 2 and its complementary ODN 3 having dC or dmC at the target site, and a rapid NO transfer reaction was observed. In contrast, a transfer reaction was not observed either with ODN 3 having dT, dA, or dG at the target site or with ODN 5-7 having dC at a nontarget site. In the analysis of deaminated products of the NO-transferred ODN 4, it was found that the transformation ratio from dmC to dT was as high as 42% together with the dmC-diazoate (13%). In conclusion, we have demonstrated the innovative method of sequence- and base-specific delivery of nitric oxide to cytidine and 5-methylcytidine. The selectivity and efficiency of NO transfer followed by deamination exhibited in this study are extremely high compared to those of the conventional methods.     

Changes of conductance and compressibility of bilayer lipid membranes induced by oligonucleotide-cationic polyene antibiotic complexes

The positively charged polyene molecule amphotericin B 3-dimethylaminopropylamide (AMA) is an efficient agent for the delivery of antisense oligodeoxyribonucleotides (ODN) into target cells. In the present study, bilayer lipid membrane (BLM) conductance, elasticity modulus perpendicular to the membrane plane, surface potential and electrical capacitance were measured by conductance and electrostriction methods in the presence of AMA, pure or complexed to 20-mer single stranded ODN at different ratios. Pure AMA did not induce changes in conductance of cholesterol-containing BLM, but did induce an increase in elasticity modulus and surface potential. ODN/AMA complexes changed BLM properties depending on the charge ratio. The most pronounced effect on membrane conductance was observed for positively charged ODN/AMA complexes (charge ratio rho-/+=0.1), while for negatively charged complexes these changes were less marked/apparent, correlating to substantially lower binding constants. The effect of ODN/AMA complexes on elasticity modulus and charge potential was biphasic. After an increase in both values, a decrease was observed for higher incubation times and ODN/AMA concentrations. These results are interpreted as indicating that the membrane property changes result from the large AMA aggregates induced by the presence of the negatively charged ODN, which condensate on these aggregates. It is suggested that the decrease of elasticity modulus and surface potential in the presence of increasing incubation time and AMA concentration result from desorption of the complexes in the complex-free compartment of the BLM cell, or appearance of a non-linear conductance of the lipid bilayer. The first alternative would explain the AMA-induced transmembrane transfer of ODN.     

C2-substituted 8-aza-7-deaza-2′-deoxyadenosines as environmentally sensitive fluorescent nucleosides for discriminating apurinic/apyrimidinic sites in DNA duplex

We synthesized various C2-naphthylethynylated 8-aza-7-deaza-2′-deoxyadenosines 1a–1c as novel environmentally sensitive fluorescent (ESF) purine nucleosides. In particular, the N,N-dimethylamino-substituted derivative 1c exhibits a remarkably high solvatochromicity (Δ$\mathrm{\lambda }$ = 111 nm). 1c-containing oligodeoxynucleotide (ODN) probes clearly discriminated an apurinic/apyrimidinic site (AP site) in the complementary strand via a change in emission wavelength and intensity when hybridized with a target ODN.     

Syntheses of 5-formyl- and 5-carboxyl-dC containing DNA oligos as potential oxidation products of 5-hydroxymethylcytosine in DNA

To investigate the potential oxidation products of 5-hydroxymethylcytosine (5-hmC)-containing DNA, we present here efficient syntheses of 5-formyl- and 5-methoxycarbonyl-2'-deoxycytidine phosphoramidites. The 5-formyl group in III was easy to introduce and was compatible with phosphoramidite and DNA syntheses. An additional treatment of ODN1 with NaBH(4) produced the corresponding ODN2 quantitatively. Phosphoramidite V was also incorporated into DNA, and the methyl ester could be hydrolyzed under mild basic conditions to afford ODN3.     

Hydrolysis of oligodeoxynucleotide phosphodiester linkages

The hydrolysis of 26-mer oligodeoxynucleotide (26-mer ODN) by cerium ions is reported. The process was analyzed by electrophoresis and the surface enhanced Raman scattering spectroscopy (SERS). Ce3+ could be oxidized to Ce4+ in oxygen atmosphere, and only Ce4+ could be used in the cleavage of ODN. We systematically studied the hydrolysis of ODN in various conditions.     

A light-controlled reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil

We describe a light-controlled template-directed reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil. Carbazole-tethered 5-carboxyvinyl-2'-deoxyuridine-containing oligodeoxynucleotide (ODN) can be ligated by irradiation at 366 nm in the presence of template ODN, and the ligated ODN can be split by irradiation at 366 nm in the absence of template ODN.     

A Quartz Crystal Microbalance (QCM) Study of Single-Strand DNA Hybridization and Hydrolytic Cleavage

The use of a quartz crystal microbalance (QCM) for monitoring in situ the immobilization of single-strand DNA marked with mercaptol group at the 5′-end on the surface of a gold-filled 7.995 MHz AT-cut quartz crystal by Au-S bond with the self-assembly technique is reported. The hybridization of ssDNA with complementary 10-mer ODN and 8-mer ODN is described. The QCM was also employed to analyze DNA cleavage by cerium(IV) ions under moderate conditions. The results showed that the QCM, which is capable of sensitive measurement, was able to investigate the immobilization, hybridization, and cleavage of ssDNA in situ. The cerium(IV) ions produced no cleavage in double-strand DNA; they were, however, able to hydrolyze single-strand DNA. Thus, the hydrolytic cleavage of ssDNA at a specific site could be ensured by protective hybridization.__________From Zhurnal Analiticheskoi Khimii, Vol. 60, No. 8, 2005, pp. 877–880.Original English Text Copyright © 2005 by Zhu, Gao, Shen, Yang, Yuan.The text was submitted by the authors in English.