Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

Chemically induced hairpin formation in DNA monolayers

A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.     

Sequence-specific control of azobenzene assemblies by molecular recognition of DNA

We investigated the molecular recognition between the amphiphile AzoAde, which is composed of azobenzene in the hydrophobic and adenine in the hydrophilic portion of the molecule, and oligonucleotides having a homogeneous base (dA30, dT30, dG30, and dC30) at the air-water interface. On the basis of the complementary base-pairing of DNA in the duplex, orderly arrangement of AzoAde on templated dT30 was examined using pi-A isotherm, UV-vis RAS, FT-IR RAS, and XPS measurements. Although there was little interaction between AzoAde and mismatched oligonucleotides (dA30, dG30, and dC30), AzoAde prepared on a dT30 subphase stoichiometrically assembled and interacted with dT30, subsequently forming a J-form assembly at the air-water interface. AFM observation of the LB films revealed the nanostructure of the J-formed AzoAde monolayer on the dT30 subphase as well as the domain structures of the H-formed monolayers on the other oligonucleotide subphases. Therefore, dT30 has a potential application as a template for assembling AzoAde at the air-water interface.     

Generalized RNA-directed recombination of RNA

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.     

Elastic Fiber-Associated Proteins of Skin in Development and Photoaging

Abstract— We sought to use antibodies against structural (tropoelastin, fibrillin) and nonstructural (decay-accelerating factor [DAF], serum amyloid P [SAP]) components of elastic fibers to characterize fiber structure in neonatal skin, normal adult skin and adult skin with solar elastosis from advanced photoaging. We found by immunohisto-chemistry and by western blotting that DAF, unlike SAP, is present on cutaneous elastic fibers in neonates and young children, suggesting that DAF may play an early, integral role in protecting elastic fibers from destruction by complement. The most superficial portion of oxytalan fibers stained with antibodies against fibrillin and DAF, while anti-tropoelastin stained only the deeper portion of oxytalan fibers. This suggests that deep oxytalan fibers are composed of both elastin and microfibrils, while the most superficial component is composed solely of microfibrillar proteins. Solar elastosis showed increased fibrillin, DAF, tropoelastin and SAP. Thus, solar elastosis is composed of both microfibrillar and elastin proteins.     

A needle-and-thread approach to bilayer transport: permeation of a molecular umbrella-oligonucleotide conjugate across a phospholipid membrane

A di-walled molecular umbrella, composed of two choloyl groups, one spermidine moiety, and a 5-thiol(2-nitrobenzoyl) "handle", was covalently attached to a 16-mer oligonucleotide (S-dT16) through a disulfide bond. Incubation of this conjugate (1) with vesicles made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (95/5, mol/mol) (200 nm diameter, extrusion) that contained entrapped glutathione (GSH) at 37 degrees C resulted in the liberation of the oligonucleotide and the umbrella-bound 5-mercapto(2-nitrobenzoyl) moiety (USH) via thiolate-disulfide interchange. The appearance of USH, together with the absence of leakage of entrapped GSH and a vesicular capture of the oligonucleotide that matches the extent of USH formation, provides compelling evidence for delivery of S-dT16 into the aqueous compartment of these vesicles. In a sense, the molecular umbrella functions like a "needle" in providing a pathway for the oligonucleotide (the "thread") to cross the membrane.     

DNA-Based Assembly of Gold Nanocrystals

Specific, designed, nonperiodic arrangements of gold nanocrystals that are 5 and 10 nm in diameter can be prepared with double-stranded DNA serving as a template (see drawing; A′ and B′ denote oligonucleotide sequences complementary to sequences A and B). The methods described should be applicable to nanocrystals composed of various materials.     

Structural and kinetic characterization of an acyl transferase ribozyme

We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.     

A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.     

Interfacing Synthetic DNA Logic Operations with Protein Outputs

DNA logic gates are devices composed entirely of DNA that perform Boolean logic operations on one or more oligonucleotide inputs. Typical outputs of DNA logic gates are oligonucleotides or fluorescent signals. Direct activation of protein function has not been engineered as an output of a DNA‐based computational circuit. Explicit control of protein activation enables the immediate triggering of enzyme function and could yield DNA computation outputs that are otherwise difficult to generate. By using zinc‐finger proteins, AND, OR, and NOR logic gates were created that respond to short oligonucleotide inputs and lead to the activation or deactivation of a split‐luciferase enzyme. The gate designs are simple and modular, thus enabling integration with larger multigate circuits, and the modular structure gives flexibility in the choice of protein output. The gates were also modified with translator circuits to provide protein activation in response to microRNA inputs as potential cellular cancer markers.     

Software note: using probe secondary structure information to enhance Affymetrix GeneChip background estimates

High-density short oligonucleotide microarrays are a primary research tool for assessing global gene expression. Background noise on microarrays comprises a significant portion of the measured raw data. A number of statistical techniques have been developed to correct for this background noise. Here, we demonstrate that probe minimum folding energy and structure can be used to enhance a previously existing model for background noise correction. We estimate that probe secondary structure accounts for up to 3% of all variation on Affymetrix microarrays.     

On-chip chemiluminescent signal enhancement using nanostructured gold-modified carbon microarrays

An original method for the enhancement of chemiluminescent (CL) on-chip detection of protein and oligonucleotides is presented. This enhancement is based on the electrodeposition of a gold nanostructured layer onto a screen-printed (SP) carbon microarray prior to the immobilization of biomolecules through a well-established diazonium adduct electrodeposition. Morphological studies of the Au layer (optical and atomic force microscopy) show that the metal film is composed of nanostructured 800 nm diameter particles covering the entire graphite surface and yielding a high surface area. Using these modified SP microarrays, enhancement factors of 229 and 126 were obtained for prostate-specific antigen (PSA) and p53 oligonucleotide detection, respectively. These enhancements were associated with three different phenomena: an enhancement of the catalyzed chemiluminescent reaction by the gold surface, an increase of the specific surface area for immobilization of the probe biomolecules, and an opposite quenching effect due to the overlapping of the gold absorption and CL emission peaks. For free PSA and target oligonucleotide detection, enhanced performances were obtained, giving detection limits of 5 ng/mL and 0.1 nM, respectively.     

A mixed film composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) brushes to enhance selectivity for detection of single nucleotide polymorphisms

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (ΔT_m) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1-0.6 M). Increases of ΔT_m when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.     

The Quantitative Determination of Hydroperoxides,Alcohols, and Ketones in Hydrocarbon Solvents

Abstract

Iodometric titration with the dead stop endpoint was used to determine the amount of alkylhydroperoxides in an oxidation reaction mixture composed of the hydrocarbon starting material, an alcohol, a ketone and an alkylhydroperoxide. Because alkylhydroperoxides decompose on glpc analysis, glpc could not be used for total analysis of the reaction mixture nor for the determination of the in situ alcohol content. However, the addition of triphenylphosphine to a separate portion of the sample quantitatively produced the corresponding alcohol from the hydroperoxide without interfering with the ketone concentration. The total alcohol content of this portion could then be determined by glpc, and the initial alcohol content found by subtracting the amount of hydroperoxide from the total alcohol.     

Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set

We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.     

Sonochemical synthesis of magnetic Janus nanoparticles

The sonochemical synthesis of nanosized surface-dissymmetrical (Janus) particles is described. The Janus particles were composed of silica and polystyrene, with the polystyrene portion loaded with nanosized magnetite particles. It is shown that the Janus particles can be used to form kinetically stable oil-in-water emulsions that can be spontaneously broken on application of an external magnetic field. The one-pot synthetic process used to prepare the Janus particles has several advantages over other conventional methods of producing such particles.     

Pentopyranosyl Oligonucleotide Systems. 9th Communication

Pyranosyl‐RNA (‘p‐RNA’ ) is an oligonucleotide system isomeric to natural RNA and composed of the very same building blocks as RNA. Its generational, chemical, and informational properties are deemed to be those of an alternative nucleic acid system that could have been a candidate in Nature's evolutionary choice of the molecular basis of genetic function. We consider the study of the chemistry of p‐RNA as etiologically relevant in the sense that knowledge of its structural, chemical, and informational properties on the chemical level offers both a perspective and reference points for the recognition of specific structural assets of the RNA structure that made it the (supposedly) superior system among possible alternatives and, therefore, the system that became part of biology as we know it today. The paper describes the chemical synthesis of β‐d‐ (and L )‐ribopyranosyl‐(4′→2′)‐oligonucleotide sequences, presents a resume of their structural and chemical properties, and cautiously discusses what we may and may not have learned from the pyranosyl isomer of RNA with respect to the conundrum of RNA's origin.     

Phospholipid membranes decorated by cholesterol-based oligonucleotides as soft hybrid nanostructures

DNA monomers and oligomers are currently showing great promise as building blocks for supramolecular arrays that can self-assemble in a fashion preprogrammed by the base pairing code. The design and build-up of hybrid DNA/amphiphilic self-assemblies can expand the range of possible architectures and enhance the selectivity toward a well-specified geometry. We report on the self-assembly properties in aqueous solution of a cholesteryl-tetraethylenglycol single stranded 18-mer oligonucleotide (ON 1TEG-Chol) and on its spontaneous insertion in fluid phospholipid membranes. Up to 500 units of these lipophilic ss-oligonucleotides can be incorporated in the outer leaflet of 350 A radius POPC vesicle. The insertion and hybridization with the complementary oligonucleotide are monitored through light scattering as an increase of hydrodynamic thickness, which is interpreted in terms of average distance between anchoring sites. The conformation of the ss-oligonucleotidic portion is strongly dependent on surface coverage, passing from a quasi-random coil to a more rigid configuration, as concentration increases. Interestingly, conformational details affect in a straightforward fashion the hybridization kinetics. Liposomes with single- and double-strand decorations remain stable within the experimental time window (about one week). The structure represents an example of successful and stable amphiphile/DNA supramolecular hybrid, where a DNA guest is held in a membrane by hydrophobic interactions. The lipophilic oligonucleotide under investigation is therefore a suitable building block that can effectively serve as a hydrophobic anchor in the fluid bilayer to assemble supramolecular constructs based on the DNA digital code.     

Ribozyme mimics as catalytic antisense reagents

Viral and fungal infections and some cancers may be described as diseases that are characterized by the expression of certain unwanted proteins. They could be termed induced genetic disorders, with induction provided by mutation or infection. A comprehensive method to inactivate injurious genes based on their nucleic acid sequences has the potential to provide effective antiviral and anticancer agents with greatly reduced side effects. We describe a chemical approach to such gene-specific pharmaceutical agents. Our initial efforts have been to develop new chemical reagents that can carry out catalytic destruction of specific mRNA sequences. We chose hydrolysis as a chemical means of destruction, because hydrolysis is compatible with living cells. Our sequence-specific catalytic RNA hydrolysis reagents may be described as functional ribozyme mimics. Reactivity is provided by small-molecule catalysts, such as metal complexes. Specificity is provided by oligonucleotide probes. Here we report initial results on the sequence-specific, hydrolytic cleavage of mRNA from the HIVgag gene, using a ribozyme mimic. The reagent is composed of a terpyridylCu(II) complex for cleavage activity and an oligonucleotide for sequence specificity.     

Oxygen-diffusion characteristics of loosely-sintered polycrystalline MgO

Self-diffusion coefficients of oxygen in well-sintered and in loosely-sintered polycrystalline MgO have been measured at an ambient oxygen pressure of about 35 mm Hg over the temperature range 1020–1450°C. The oxygen diffusion coefficient as a function of temperature of the former specimen is expressed by a single Arrhenius equation with an activation energy of 55.8 kcal/mole, while that of the latter is composed of two intersecting lines (E = 60.2 kcal/mole from 1020–1250°C; E = 102.8 kcal/mole from 1250–1450°C). The diffusivity for single-crystal grain of the well-sintered polycrystals is two orders of magnitude larger than that in the low-temperature portion of the loosely-sintered one. This fact is interpreted in terms of an insufficient dissolution of monovalent cation impurities into the loosely-sintered crystal lattice. Two ways of interpretation are given for the presence of the high-temperature portion characteristic of the loosely-sintered polycrystal.