Multi-charged oligonucleotide ion formation in sonic spray ionization.

An oligonucleotide tends to release hydrogen atoms from a phosphoric acid group and to form negative ions that can be detected by mass spectrometry. Usually, with a solution-spray based ionization technique, the negative ions are present in different charge states. Ion formation for the nucleotide is quite complicated and is easily influenced by matrix and other constituents in a sample solution, as well as by the operating parameters for a mass spectrometer. In this work, we studied oligonucleotide ion formation by using an ion trap mass spectrometer combined with a sonic spray ionization (SSI) source. An oligonucleotide with 20 bases was measured. Effects from contaminants and parameters affecting the ion production, such as a high voltage applied to the ionization source and sample solution-flow rate, were investigated. Our results showed that an ion with about one charge for every three bases was most abundant. However, the signal intensity and the mass spectrum pattern were sensitive to the matrix and operating parameters. One of the reasons for such sensitivity is that there are various ion states for an oligonucleotide. Any change in the matrix or an operating parameter may shift the balances between the ion states. Adding Tris, or (hydroxymethyl)aminomethane, enhanced the signal intensity of the oligonucleotide and promoted formation of the oligonucleotide ion with higher charges, while adding acetic acid favored the ions with lower charges, compared with that obtained in the medium without adding Tris and acetic acid. The effects on charged droplets and chemical enhancement were investigated. The mechanism for oligonucleotide ion formation is discussed.     

Electrochemical response of oligonucleotides on carbon paste electrode

Electrochemical response of synthetic oligonucleotides with different DNA bases sequences was investigated to find relationships between a chain composition and a signal. All DNA mononucleotides present electroactivity at a carbon paste electrode yielding anodic peaks at potentials: 1.00 (GMP), 1.28 (AMP), 1.47 (TMP) and 1.53 V (CMP). Also 15-mer homooligonucleotides show respective anodic peaks. Electrochemical response of 15- and 19-mer oligonucleotides consisting of all four DNA bases in different amounts was determined by the composition of oligonucleotide chain. When the contribution of different bases in oligonucleotide was balanced two anodic peaks were obtained that can be attributed to guanine and adenine moieties. Thymine residue is shown as a separate peak in voltammogram when its content in oligonucleotide chain is close to 50% of the total number of bases. Cytosine also yields a peak at its significant contribution in oligonucleotide chain and both pyrimidinic moieties produce catalytic waves easier when one of them is dominating or when only one pyrimidine derivative is present in a chain. Guanine is the easiest oxidized base and it produces a peak even at its minimal contribution (one guanine residue in 19-mer oligonucleotide). Guanine peak potential is dependent on oligonucleotide concentration and oligonucleotide composition. The lowest oligonucleotide concentration detected by guanine peak was 12.5 nM whereas detected by thymine peak was 90 nM.     

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.     

Cationic oligonucleotide-peptide conjugates with aggregating properties enter efficiently into cells while maintaining hybridization properties and enzymatic recognition

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems. Moreover, stability of the duplex formed with the complementary sequence increased without inhibiting oligonucleotide enzyme recognition as shown by the properties of the conjugate to prime chain elongation by Taq DNA polymerase in a linear amplification/sequencing process.     

Using sol-gel/crown ether hybrid materials as desalting substrates for matrix-assisted laser desorption/ionization analysis of oligonucleotides

This study demonstrates the feasibility of using sol-gel/crown ether hybrid materials as sample substrates that reduce the intensity of the signals of sodium ion adducts of oligonucleotides during matrix-assisted laser desorption/ionization (MALDI) analysis. 2-Hydroxymethyl[15]crown-5 and 2-hydroxymethyl[18]crown-6 were added as dopants during the sol-gel process to generate desalting substrates for MALDI sample deposition. The results demonstrate that the sol-gel/crown ether hybrid materials effectively suppress the formation of sodiated oligonucleotides during MALDI analysis. The largest detectable molecular size for an oligonucleotide was a 100-mer, and the detection limit for an oligonucleotide 36-mer was ca. 20 fmol.     

Guanine-thymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication

An intrastrand cross-link lesion, in which two neighboring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequence oligonucleotides in order to perform in vitro replication studies using either bacterial replicative or translesional synthesis polymerases. The investigated tandem base lesion that involves a cross-link between the methylene group of thymine and the C8 of an adjacent guanine residue has been prepared by UV-photolysis under anaerobic condition of the photolabile precursor 5-(phenylthiomethyl)-2'-deoxyuridine that has been site-specifically incorporated into a 9-mer oligonucleotide. After ligation, the lesion-containing modified oligonucleotide was used as a DNA template in primer extension reactions catalyzed by several DNA polymerases including the fragment Klenow exo-(Kf-) of E. coli polymerase I, the Thermus aquaticus polymerase (Taq pol) and the E. coli translesional DNA polymerase Pol IV (dinB). It was found that the primer extension reaction was stopped after the incorporation of the correct nucleotide dAMP opposite the 3'-thymine residue of guanine(C8-CH2) thymine lesion by Kf- and Pol IV; however it was noted that the efficiency of the nucleotide incorporation was reduced. In contrast, the Taq polymerase was totally blocked at the nucleotide preceding the tandem lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for prokaryotic DNA polymerases, being likely lethal for the cell.     

Benzofurazane as a New Redox Label for Electrochemical Detection of DNA: Towards Multipotential Redox Coding of DNA Bases

Benzofurazane has been attached to nucleosides and dNTPs, either directly or through an acetylene linker, as a new redox label for electrochemical analysis of nucleotide sequences. Primer extension incorporation of the benzofurazane‐modified dNTPs by polymerases has been developed for the construction of labeled oligonucleotide probes. In combination with nitrophenyl and aminophenyl labels, we have successfully developed a three‐potential coding of DNA bases and have explored the relevant electrochemical potentials. The combination of benzofurazane and nitrophenyl reducible labels has proved to be excellent for ratiometric analysis of nucleotide sequences and is suitable for bioanalytical applications.     

Towards a target label-free suboptimum oligonucleotide displacement-based detection system

A novel method for the future development of label-free DNA sensors is proposed here. The approach is based on the displacement of a labelled suboptimum mutated oligonucleotide hybridised with the immobilised biotin-capture probe. The target fully complementary to the biotin-capture probe can displace the labelled oligonucleotide causing a subsequent decrease of the signal that verifies the presence of the target. The decrease of signal was demonstrated to be proportional to the target concentration. A study of the hybridisation of mutated and complementary labelled oligonucleotides with an immobilised biotin-capture probe was carried out. Different kinetic and thermodynamic behaviour was observed for heterogeneous hybridisation of biotin-capture probe with complementary or suboptimum oligonucleotides. The displacement method evaluated colourimetrically achieved the objective of decreasing the response time from 1 h for direct hybridisation of 19-mer oligonucleotides in the direct enzyme-linked oligonucleotide assay (ELONA) to 5 min in the case of displacement detection in the micromolar concentration range. Figure The detection system is based on the displacement of suboptimum HRP-labelled mutated oligonucleotide by the fully complementary target     

Optical DNA detection based on gold nanorods aggregation

Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Herein, the localized surface plasmon resonance properties of unmodified gold nanorods (GNRs) in 1 mM cetyltrimethyl-ammonium bromide solution were used for sensing DNA sequences, with good simplicity and sensitivity. The intensity of typical plasmon resonance absorption bands of the GNRs decreased with increasing cDNA concentration. The detection of a 30-mer single-stranded oligonucleotide as a model reached a detection limit of about 0.1 pM. This study will be significant for as-prepared GNRs for future application in biological systems.     

LNA-modified oligodeoxynucleotide hybridization with DNA microarrays printed on nanoporous membrane slides

We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to approximately 4 nL with detectable signal intensity. Moreover, the amount of the DNA target could be reduced to 4 fmol. The described approach could dramatically increase the throughput of techniques based on sequencing by hybridization, such as oligofingerprinting, by decreasing the total number of probes that are needed for analysis of large clone sets and reduction of the sample/reagent consumption. The method is particularly advantageous when numerous hybridization-based assays must be performed for characterization of sample sets of 100,000 or more.     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

高灵敏纳米金比色芯片检测乙型肝炎病毒DNA及其变异

构建了新型纳米金比色芯片,利用Taq DNA连接酶的连接特异性,将其与乙型肝炎病毒DNA( HBV-DNA)靶序列完全互补杂交的捕获探针(固定在芯片上)和纳米金修饰的探针连接成一条链,从而将纳米金颗粒固定到芯片点阵上,再通过银染反应放大,形成裸眼可见的显色信息.通过点阵的位置及灰度,即可判断HBV-DNA靶序列的单碱基突变,并得出相对定量信息.本实验对不同浓度的HBV-DNA靶序列进行了检测.结果显示:此技术对单碱基突变有很强的特异性识别能力,并且具有较高的灵敏度(约10 pmol/L),在10～100 pmol/L浓度范围内表现出较好的线性关系.该技术检测时间短(＜1 h)、操作简单、不需要特殊的检测设备,具有很好的临床应用前景.     

Electrochemical detection of a Vibrio parahaemolyticus sequence-specific gene based on a gold electrode modified with a single stranded probe

An electrochemical DNA biosensor for specific-sequences detection of Vibrio parahaemolyticus (VP) was fabricated. A single-stranded 20-mer oligonucleotide (ssDNA) and 6-mercapto-1-hexanol (MCH) were immobilized via a thiol linker on gold disk electrodes by self-assembling. The ssDNA underwent hybridization in a hybridization solution containing complementary or non-complementary or single base pair mismatched DNA sequences of VP. Examination of changes in response to these three target DNAs showed that the developed biosensor had a high selectivity and sensitivity.     

Sequence-selective DNA cleavage by a chimeric metallopeptide

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.     

Amperometric detection of DNA hybridization on a gold surface depends on the orientation of oligonucleotide chains

We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.     

Application of DNA-binding polymers for preparation of DNA for analysis by matrix-assisted laser desorption/ionization mass spectrometry

The susceptibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the presence of salts in a sample, especially salts of alkali metals, requires careful and often tedious desalting procedures which complicate and slow the throughput of MS-based methods. A novel approach to sample preparation was developed based on the extraction of DNA out of solution onto a solid surface with an attached DNA-binding polymer, such as polyethyleneimine or polyvinylpyrrolidone. The observed binding is strong enough to sustain washing, and, as a result, desalting and concentration can be performed in a single fast step. After DNA has been immobilized on the surface and supernatant solution removed, subsequent addition of MALDI matrix releases material from the surface, which co-crystallizes with matrix. The mass spectrometric analysis is then performed directly from this support. Analysis of oligonucleotides and three-fold multiplexed SNP typing reactions performed by this method shows improved sensitivity and excellent resolution for various DNA fragments, together with high tolerance to various buffer components, such as alkali metals and surfactants. Simplicity and speed make it attractive for high-throughput sample preparation and analysis of oligonucleotide mixtures by MALDI-MS.     

功能化纳米金放大的DNA电化学传感器研究

研究了DNA夹心杂交和直接杂交体系,将功能化纳米金引入到标记有生物素的杂交双链上,制成具有电化学活性和纳米金放大作用的DNA电化学传感器,采用循环伏安法测试.在夹心杂交体系中,靶点DNA浓度与阳极峰电流关系曲线的相对标准偏差为3.0%～13.0%,在浓度为6.9×10^-3～0.14nmol/L范围内得到良好的线性关系,检测限达到2.0×10^-3nmol/L,实现了对单碱基突变的高灵敏检测和序列识别.直接杂交检测限为2.5×10^-4mol/L,分别在2.5×10^-4～5.0×10^-3nmol/L和5.0×10^-3～10nmol/L范围内得到峰电量与浓度的良好线性关系.并比较这两种体系.     

Label-free electrochemical detection of DNA hybridization on gold electrode

A label-free electrochemical detection protocol for DNA hybridization is reported for the first time by using a gold electrode (AuE). The oxidation signal of guanine was monitored at +0.73 V by using square wave voltammetry (SWV) on self-assembled l-cysteine monolayer (SAM) modified AuE. The electrochemical determination of hybridization between an inosine substituted capture probe and native target DNA was also accomplished. 6-mer adenine probe was covalently attached to SAM via its amino link at 5^′ end. Then, 6-mer thymine-tag of the capture probe was hybridized with the adenine probe, thus left the rest of the oligonucleotide available for hybridization with the target. The dependence of the guanine signal upon the concentration of the target was observed. Probe modified AuE was also challenged with non-complementary and mismatch containing oligonucletides. Label-free detection of hybridization on AuE is greatly advantageous over the existing carbon and mercury electrode materials, because of its potential applicability to microfabrication techniques. Performance characteristics of the genosensor are described, along with future prospects.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Synthesis,incorporation into triplex-forming oligonucleotide,and binding properties of a novel 2'-deoxy-C-nucleoside featuring a 6-(thiazolyl-5)benzimidazole nucleobase

[reaction: see text] 6-(Thiazolyl-5)benzimidazole (B(t)()) was designed as a novel nucleobase for the specific recognition of an inverted A.T base pair in a triple helix motif. It was successfully incorporated into an 18-mer triplex-forming oligonucleotide (TFO) using the 2'-deoxy-C-nucleoside phosphoramidite 16. The triple helix binding properties of the modified TFO were examined by means of thermal denaturation experiments targeting an oligopyrimidine.oligopurine 26-mer DNA duplex containing an A.T base pair inversion.