Oligodeoxynucleotide Probes Research Articles

Remarkable acceleration of a DNA/RNA inter-strand functionality transfer reaction to modify a cytosine residue: the proximity effect via complexation with a metal cation.

Modified nucleosides in natural RNA molecules are essential for their functions. Non-natural nucleoside analogues have been introduced into RNA to manipulate its structure and function. We have recently developed a new strategy for the in situ modification of RNA based on the functionality transfer reaction between an oligodeoxynucleotide probe and an RNA substrate. 2′-Deoxy-6-thioguanosine (6-thio-dG) was used as the platform to anchor the transfer group. In this study, a pyridinyl vinyl ketone moiety was newly designed as the transfer group with the expectation that a metal cation would form a chelate complex with the pyridinyl-2-keto group. It was demonstrated that the (E)-pyridinyl vinyl keto group was efficiently and specifically transferred to the 4-amino group of the opposing cytosine in RNA in the presence of NiCl2 with more than 200-fold accelerated rate compared with the previous system with the use of the diketo transfer group. Detailed mechanistic studies suggested that NiCl2 forms a bridging complex between the pyridinyl keto moiety and the N7 of the purine residue neighboring the cytosine residue of the RNA substrate to bring the groups in close proximity.

Scanning Electrochemical Microscopy of DNA Hybridization on DNA Microarrays Enhanced by HRP-Modified SiO2 Nanoparticles

Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe via sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO2 nanoparticles through the biotin-streptavidin interaction. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10(-7)-10(-12) M). This technique may find applications in genomic sequencing.

Detection of mRNA of the Cyclin D1 Breast Cancer Marker by a Novel Duplex-DNA Probe

Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6, as a fluorescent uridine analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red-shifted as compared to uridine. Here, we utilized 6 for preparation of labeled oligodeoxynucleotide (ODN) probes based on MS2 and cyclin D1 (a known breast cancer mRNA marker) sequences. Cyclin D1-derived labeled-ssODN showed a 9.5-fold decrease of quantum yield upon duplex formation. On the basis of this finding, we developed the ds-NIF (nucleoside with intrinsic fluorescence)-probe methodology for detection of cyclin D1 mRNA, by which the fluorescent probe is released upon recognition of target mRNA by the relatively dark NIF-duplex-probe. Indeed, we successfully detected, a ss-deoxynucleic acid (DNA) variant of cyclin D1 mRNA using a dark NIF-labeled duplex-probe, and monitoring the recognition process by fluorescence spectroscopy and gel electrophoresis. Furthermore, we successfully detected cyclin D1 mRNA in RNA extracted from cancerous human cells, using ds-NIF methodology.

Qualitative and quantitative detection of DNA amplified with HRP-modified SiO2 nanoparticles using scanning electrochemical microscopy

Qualitative and quantitative detection of DNA was achieved by a “sandwich” DNA sensor through SG/TC (substrate generation and tip collection) mode of scanning electrochemical microscopy (SECM). The “sandwich” DNA structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture probe), assembled on the gold substrate surface, with target DNA and biotinylated indicator probe. HRP (horseradish peroxidase)-wrapped SiO2 nanoparticles were linked to the sandwich structure through biotin–streptavidin interaction. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface where sequence-specific hybridization had occurred through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. For SECM imaging experiment, we structured the microsensor platform through localized desorption of 1-dodecanethiol monolayer. Approach curves were employed for quantitative detection of DNA concentration. The detection limit of complementary DNA was as low as 0.8pM. This technique is promising for the application on electrochemical DNA chip.

Polyvinyl Pyrrolidone Promotes DNA Cleavage by a ROS-Independent and Depurination Mechanism

Polyvinyl pyrrolidone polymer (PVP) has been widely applied in biological and medical fields. A few in vitro studies indicated that PVP might cause toxicity. However, the underlying mechanism is poorly understood. In this work, we found that PVP directly induced strand breakages of various DNA molecules, implicating a cleavage activity. Moreover, reactive oxygen species (ROS) scavenging analysis shows that DNA cleavage activity of PVP is not related to ROS-induced oxidation. As revealed by gel electrophoresis and liquid chromatography/mass spectrometry analysis, the major cleavage products of DNA were identified as two purine bases, guanine and adenine, suggesting that PVPs have a novel depurination activity. The selective depurination and DNA cleavage activity of PVPs were further confirmed by studying the interaction of PVP with four nucleosides and four well-designed oligodeoxynucleotides probes containing specific nucleotides. This study may provide insights into PVP-DNA interactions and resultant genotoxicity and may also open a new way for DNA study.

Selective Individual Primary Cell Capture Using Locally Bio-Functionalized Micropores

BackgroundSolid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy.Methodology/Principal FindingsWe locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins.Conclusions/SignificanceThe results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures.

ChemInform Abstract: The Oligodeoxynucleotide Probes for the Site‐Specific Modification of RNA

AbstractReview: 52 refs.

Quencher-free Linear Beacon Systems with Dual Fluorene-labeled Deoxyuridines

The identification of natural genetic variation in human populations is arguably one of the most important spin-offs of the effort toward the completion of the Human Genome Project. Single nucleotide polymorphisms (SNPs) are the most abundant natural genetic variations in the human genome. To satisfy the demand for molecular diagnostic assays for identifying SNPs, Tyagi and Kramer developed fluorogenic hairpin-shaped DNA probes, called “molecular beacons in 1996. Conventional molecular beacons are single-stranded oligodeoxynucleotides (ODNs) probes that exist in solution as stable stem-and-loop structures in which a fluorophore is covalently linked to one end and a quencher is covalently linked to the other end. The stem keeps the fluorophore and quencher of the molecular beacon in extremely close proximity to each other, when they are not bound to their targets; this causes the fluorescence of the fluorophore to be quenched via the fluorescence resonance energy transfer. By contrast, the conformational change induced by binding to a target nucleic acid causes the fluorophore and the quencher to move away from each other, thereby restoring the fluorescence. Therefore, the presence of the target nucleic acid is characterized by a sharp increase in fluorescence intensity. Recently, we reported that fluorescent hairpin oligonucleotides can function as molecular beacons even without the attachment of additional quencher moieties. Such probes can be called “quencher-free molecular beacons. A quencher-free molecular beacon consists of only a single fluorophore (2-ethynylfluorene-labeled deoxyuridine, U) positioned in the hairpin loop. When this probe undergoes DNA hybridization, it can discriminate between fully matched and single-base-mismatched sequences by exhibiting a strong dA-selective fluorescence. When the fluorene unit is intercalated within a DNA duplex as a result of mismatched base pairing, the fluorescence of the U unit is quenched as a result of a photoinduced charge transfer originating from interactions with neighboring nucleobases. In particular, the quencher-free molecular beacon probe having C-flanking bases (C-FBs) shows more efficient fluorescence ON/OFF behavior than do any other combinations of FBs. The only drawback of this quencher-free molecular beacon system is its poor detection sensitivity due to its relatively high intrinsic emission intensity. To solve this problem, we recently positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite the U unit to reduce the intrinsic fluorescence on hybridization with a probe. A short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective in reducing the intrinsic fluorescence of a linear beacon. In another effort to eliminate this drawback, we employed an ODN containing dual U units at specific distances (Figure 1). We observed that using this dual-labeled linear beacon system resulted in a clear improvement in the detection sensitivity for the perfectly matched target. The modified nucleotide U was synthesized through Sonogashira coupling of a 2-ethynylfluorene unit at the 5position of a 2-deoxyuridine base. Incorporations of the fluorene-labeled deoxyuridine U into the central position of the ODN 1 and of the two U units into the positions with one deoxycytosine spacer unit were effected using standard protocols of automated DNA synthesis (Figure 2). The fluorescence properties of fluorophore-containing DNA are strongly dependent on the electron injection and transfer properties of the FBs. In particular, fluorescent ODNs having C-FBs produce efficient fluorescence ON/OFF systems and have higher sensitivity as probes than do any other combinations of FBs. Therefore, we positioned two deoxy-

Detection of single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer using a fluorophore-tagged DNA probe

Single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer were identified using oligodeoxynucleotide probe sequences bearing internal anthracene fluorophores proximal to the SNP site. Depending upon the nature of the synthesised target sequences, probe-target duplex formation could lead to enhanced or attenuated fluorescence emission from the anthracene, enabling detection of a proximal base-pair as either matching or mismatching.

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.

Intrastrand locks increase duplex stability and base pairing selectivity

Oligodeoxynucleotide probes with disulfide locks between neighboring nucleobases show increases in melting point for duplexes with RNA target strands of up to 7.6 °C. The weakly pairing TT dimers are replaced with locked 2'-deoxy-5-(thioalkynyl)uridine residues via automated synthesis.

The oligodeoxynucleotide probes for the site-specific modification of RNA

As the knowledge of the biological functions of RNA expands, the demand for research tools to investigate intracellular RNA is increasing. Oligonucleotides can be rationally designed for the target RNA sequence, and therefore, have become a reliable platform for the development of specific molecules for RNA. The chemical modification of RNA has a strong impact on RNA research; the fluorescent labeling of RNA is useful to monitor RNA production, processing, relocation in the cell, interaction with other intracellular components and degradation, etc. Chemical modification may affect the RNA function through a variety of pathways, and therefore, would be potentially useful for biological research, therapeutic approach and artificial manipulation of the RNA function. This tutorial review starts with an introduction of the biological relevance of modified RNA, and focuses on the recent progress of the oligodeoxynucleotide probes for the covalent modifications of RNA. The prospects of this new technology are also discussed.

Templated chemistry for sequence-specific fluorogenic detection of duplex DNA.

We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple-helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an α-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double-stranded DNA, at least for homopurine-homopyrimidine target sites, under native and nondenaturing conditions.

Synthesis and Biophysical Properties of Oligodeoxynucleotides Containing 2′‐Deoxy‐5‐(4‐nitro‐1H‐imidazol‐1‐yl)‐β‐D‐uridine and 2′‐Deoxy‐5‐(1,3‐dioxo‐1H‐benzo[de]isoquinolin‐2(3H)‐yl)‐β‐D‐uridine Monomers

Detection of single-nucleotide polymorphisms (SNPs) of biologically relevant DNA and RNA samples remain a scientific and practical challenge. We have synthesized phosphoramidite building blocks and oligodeoxynucleotide probes containing novel 2'-deoxyuridine monomers modified by 5-(4-nitro-1H-imidazol-1-yl; (monomer X) or 2'-deoxy-5-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl; monomer Y) substituents. The effects of monomers X and Y on duplex thermal stability, and their capability towards discrimination of single-base mismatches were furthermore studied. Encouraging results were obtained with respect to thermal mismatch discrimination using oligodeoxynucleotides containing monomer X and fluorescence-based discrimination using oligodeoxynucleotides containing monomer Y.

A new usage of functionalized oligodeoxynucleotide probe for site-specific modification of a guanine base within RNA

Site-specific modification of RNA is of great significance to investigate RNA structure, function and dynamics. Recently, we reported a new method for sequence- and cytosine-selective chemical modification of RNA based on the functional group transfer reaction of the 1-phenyl-2-methylydene-1,3-diketone unit of the 6-thioguanosine base incorporated in the oligodeoxynucleotide probe. In this study, we describe that the functionality transfer rate is greatly enhanced and the selectivity is shifted to the guanine base when the reaction is performed under alkaline conditions. Detailed investigation indicated that the 2-amino group of the enolate form of rG is the reactant of the functionality transfer reaction. As a potential application of this efficient functionality transfer reaction, a pyrene group as a relatively large fluorescent group was successfully transferred to the target guanine base of RNA with a high guanine and site selectivity. This functionality transfer reaction with high efficiency and high site-selectivity would provide a new opportunity as a unique tool for the study of RNA.

Regulation of Expression of the Rat Orthologue of Mouse Double Minute 2 (MDM2) by H2O2-induced Oxidative Stress in Neonatal Rat Cardiac Myocytes

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.

Preparation and Quality Test of Superparamagnetic Iron Oxide Labeled Antisense Oligodeoxynucleotide Probe: A Preliminary Study

Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that transfected SK-Br-3 oncocytes by antisense probe had the lowest signal of all. The SPIO-labeled ASODN probe shows unique features including well-distributed spherical morphology, high conjugating rate and loading efficiency, and the signal intensity of SPIO-labeled ASODN-transfected SK-Br-3 oncocytes is reduced in MR imaging. These results indicate that the SPIO-labeled ASODN probe is potentially useful as a MR targeting contrast enhancing agent to specifically diagnose tumors which had over-expression of the c-erbB2 oncogene at an early stage.

Cloning of the gene encoding an insecticidal protein inPseudomonas pseudoalcaligenes

Pseudomonas pseudoalcaligenes strain PPA (first isolated from yellow-spined bamboo locust,Ceracris kiangsu, in 1991) is an effective biological control agent for locust.Escherichia coli DH5α was transformed using a partialSau3AI digest ofP. pseudoalcaligenes chromosomal DNA ligated withpUC19 vector cut byBamHI. The resulting genomic library was screened using an oligodeoxynucleotide probe derived from the N-terminal sequence of the purified insecticidal protein. Five positive clones were obtained after two rounds of screening. The 1.4 kbBamHI-KpnI fragment of the smallest clone (pSC1) contained the N terminal sequence of the insecticidal protein. Further sequencing identified an open reading frame encoding a polypeptide of 276 amino acid residues that is thought to contain a signal peptide of 22 amino acids and a mature protein of 254 amino acids. The calculated molecular weight of 26.7 kDa for the putative mature protein is close to that of the native insecticidal protein. The insecticidal protein lacked significant identity with toxic proteins from other bacteria upon comparison of amino acid sequences. ABamHI-KpnI fragment was cloned intopT7-6. A novel protein was expressed inE. coli BL21 (DE3) and its western blot similar to natural toxin.

Formation of the double helix: a mutational study

To investigate the mechanisms by which oligonucleotides hybridize to target molecules, the binding of two oligodeoxynucleotide probes to RNA targets was measured over a broad range of temperatures. Mutations were then scanned across each DNA/RNA hybrid to map, at single base resolution, sequences important for hybridization. Despite being unrelated in sequence, each hybrid formed by a similar mechanism. In the absence of secondary structure, two stretches of bases, termed nucleation regions, cooperated with one another by a looping mechanism to nucleate hybridization. Mutations inside each nucleation region strongly decreased hybridization rates, even at temperatures well below the melting temperature (Tm) of the hybridized duplex. Surprisingly, nucleation regions were detected in a RNA target but not a corresponding DNA target. When either nucleation region was sequestered in secondary structure, the hybridization rate fell and the mechanism of hybridization changed. Single-stranded bases within the nucleation region of the probe and target first collided to form a double helix. If sufficiently G + C rich, the double helix then propagated throughout the oligonucleotide by a strand invasion process. On the basis of these results, general mechanisms for the hybridization of oligonucleotides to complementary and mutant targets are proposed.

A novel DNA biosensor based on ellipsometry

In this study, we have firstly investigated the effects of dipping time, solution concentration and solvent types on the formation of self-assembled monolayers (SAMs) with 3-mercaptopropyltrimethoxysilane (MPTS) molecules on the Si(0 0 1) surfaces. Ellipsometric measurements showed that monolayers with a thickness of about 0.73 nm were formed when the dipping time is about 1 h, while more profound agglomerations were observed for longer time periods and MPTS solutions with higher concentrations. Monolayers were formed with solvents having larger dielectric constants. Contact angle measurements were in good correlation with the ellipsometric data. 5′-Thiolated oligodeoxynucleotides (ODNs) probes were immobilized onto these thiol-terminated SAMs by disulfide bond formation. The thickness of the ODN-layer on the MPTS modified surfaces reached almost a constant value of about 2.5 nm in 1 h. Target ODNs were detected by monitoring hybridization onto the surfaces by ellipsometry. The analytical signal (the delta ( Δ) value) measured was correlated with the target ODNs concentration.