Oligonucleotide Chain Research Articles

Immobilization and stretching of 5'-pyrene-terminated DNA on carbon film deposited on electron microscope grid.

The immobilization and stretching of randomly coiled DNA molecules on hydrophobic carbon film is a challenging microscopic technique, which possess various applications, especially for genome sequencing. In this report the pyrenyl nucleus is used as an anchor moiety to acquire higher affinity of double stranded DNA to the graphite surface. DNA and pyrene are joined through a linker composed of four aliphatic methylene groups. For the preparation of pyrene-terminated DNA a multifunctional phosphoramidite monomer compound was designed. It contains pyrenylbutoxy group as an anchor moiety for π-stacking attachment to the carbon film, 2-cyanoethyloxy, and diisopropylamino as coupling groups for conjugation to activated oligonucleotide chain or DNA molecule. This monomer derivative was suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The successful immobilization and stretching of pyrene-terminated DNA was demonstrated by conventional 100 kV transmission electron microscope. The microscopic analysis confirmed the stretched shape of the negatively charged nucleic acid pieces on the hydrophobic carbon film.

Synthesis of C-5, C-2′ and C-4′-neomycin-conjugated triplex forming oligonucleotides and their affinity to DNA-duplexes

Neomycin-conjugated homopyrimidine oligo 2′-deoxyribonucleotides have been synthesized on a solid phase and their potential as triplex forming oligonucleotides (TFOs) with DNA-duplexes has been studied. For the synthesis of the conjugates, C-5, C-2′ and C-4′-tethered alkyne-modified nucleoside derivatives were used as an integral part of the standard automated oligonucleotide chain elongation. An azide-derived neomycin was then conjugated to the incorporated terminal alkynes by Cu(I)-catalyzed 1,3-dipolar cycloaddition (the click chemistry). Concentrated ammonia released the desired conjugates in acceptable purity and yields. The site of conjugation was expectedly important for the Hoogsteen-face recognition: C-5-conjugation showed a notable positive effect, whereas the influence of the C-2′ and C-4′-modification remained marginal. In addition to conventional characterization methods (UV- and CD-spectroscopy), 19F NMR spectroscopy was applied for the monitoring of triplex/duplex/single strand-conversions.

Characterization of oligosorbents and application to the purification of ochratoxin A from wheat extracts

The aim of this work was to optimize the preparation of an anti-ochratoxin A (OTA) oligosorbent (OS), a solid-phase extraction sorbent based on OTA aptamers covalently immobilized on sepharose. Different syntheses were carried out by modifying the side of the oligonucleotide chain bound to the sepharose, the length of the spacer arm between the aptamer and the sepharose and the amount of the aptamers introduced during the covalent grafting. Indeed, the capacity of OSs prepared using 3'- or 5'-amino-modified sequences with a C6 or a C12 was studied. In the best conditions, the concentration of aptamers sequence used during their grafting was increased and a capacity close to 40 nmol g(-1) of OS was reached. The potential of the resulting OSs was also studied in pure media. For this, their selectivity was checked by comparing them to a control sorbent prepared without immobilizing aptamers. Extraction recoveries close to 100% were obtained on all OSs, while no retention was observed on the control sorbent. OS does not demonstrate any cross-reactivity towards OTA metabolites, i.e., ochratoxin B and ochratoxin hydroquinone. The oligosorbent was finally applied to the clean-up of OTA from wheat sample extracts. Extraction recoveries were not affected by matrix interferences and the resulting chromatogram clearly highlights the selectivity of the sorbent that allows the removal of matrix components thus improving the reliability of the quantitation of OTA in real samples.

Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads® with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acids.

Synthesis of 4-(4-methoxyphenyl)-2,6-dinitrobenzaldehyde

Photoacid generators capable of undergoing decomposition under irradiation with visible light are very important for efficient oligonucleotide microarray fabrication. In this case, there are no detrimental effect of UV irradiation on growing oligonucleotide chain and related side reactions leading to degradation of targeted oligonucleotides [1, 2]. An important intermediate product in the synthesis of photoacids necessary for the preparation of oligonucleotide bioarrays is 4-(4-methoxyphenyl)-2,6-dinitrobenzaldehyde, which was previously synthesized by oxidation of 2-bromomethyl-5(4-methoxyphenyl)-1,3-dinitrobenzene (I) with bis(tetrabutylammonium) dichromate on prolonged heating in boiling chloroform; however, the yield of II was as poor as 23% [2]. The goal of the present work was to develop a procedure which could be the most convenient for the preparation of 4-(4-methoxyphenyl)2,6-dinitrobenzaldehyde. The transformation of benzyl halides into the corresponding aldehydes is widely used in synthetic organic chemistry [3–5]. Benzyl halides can be converted into aldehydes via oxidation according to Kornblum [4]. However, the Kornblum oxidation of benzyl bromide I in DMSO in the presence of NaHCO3 (Scheme 1) gave a mixture of aldehyde II and alcohol III at a ratio of ~2 : 1 (cf. [6]). By oxidation of that mixture with pyridinium chlorochromate (PCC) in methylene chloride we succeeded in obtaining aldehyde II in 90% yield. Variation of the reaction conditions made it possible to synthesize alcohol III in 94% yield from compound I, and the subsequent oxidation of III with PCC quantitatively afforded aldehyde II. In addition, benzyl halides can be converted into aldehydes according to Krohnke [5]. The reaction of pyridinium salt IV with N,N-dimethyl-4-nitrosoaniline hydrochloride and subsequent hydrolysis of Schiff ISSN 1070-4280, Russian Journal of Organic Chemistry, 2013, Vol. 49, No. 7, pp. 1089–1091. © Pleiades Publishing, Ltd., 2013. Original Russian Text © E.B. Nikolaenkova, I.A. Os’kina, V.A. Savel’ev, V.A. Samsonov, A.Ya. Tikhonov, V.A. Ryabinin, A.N. Sinyakov, 2013, published in Zhurnal Organicheskoi Khimii, 2013, Vol. 49, No. 7, pp. 1103–1105.

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver-directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi-functional targeting molecule, which maintains targeting, endosome-escaping, and DNA-binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full-length cDNA encoding anti-hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50-Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC-C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv-Mel-Gal4 triple fusion protein (C1MG) was purified with a Ni(2+) chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni(2+) column purification. Western blot and immunohistochemistry showed the antigen-binding ability of C1MG to the cell surface of the liver-derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane-disrupting activity. DNA-binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome-escaping and DNA-binding capacity, which makes it probable to further study its liver-specific DNA delivery efficacy in vivo.

2′-modified oligoribonucleotides containing 1,2-diol and aldehyde groups. Synthesis and properties

1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.

Solid-Supported NOTA and DOTA Chelators Useful for the Synthesis of 3′-Radiometalated Oligonucleotides

Esterified precursors of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; 18) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA; 17,19) ligands bearing a dimethoxytritylated hydroxyl side arm were prepared and immobilized via an ester linkage to long chain alkyl amine derivatized controlled pore glass (LCAA-CPG). Oligonucleotide chains were then assembled on the hydroxyl function and conjugates were released and deprotected by a two-step cleavage with aqueous alkali and ammonia. The 3'-DOTA and 3'-NOTA conjugated oligonucleotides were converted to (68)Ga chelates by a brief treatment with [(68)Ga]Cl(3) at elevated temperature. Applicability of the conjugates for in vivo imaging with positron emission tomography (PET) was verified.

A Universal Nucleoside with Strong Two-Band Switchable Fluorescence and Sensitivity to the Environment for Investigating DNA Interactions

With the aim of developing a new tool to investigate DNA interactions, a nucleoside analogue incorporating a 3-hydroxychromone (3HC) fluorophore as a nucleobase mimic was synthesized and incorporated into oligonucleotide chains. In comparison with existing fluorescent nucleoside analogues, this dye features exceptional environmental sensitivity switching between two well-resolved fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard. Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-selectively monitored.

DNA-porphyrin hybrids as reaction centers for photosensitized ene reactions with singlet oxygen

Recent developments in DNA-mediated nanostructuring have paved the way for the development of novel reaction centers. As part of a project focused on nanostructured reaction centers for reactions catalyzed by porphyrins, we have developed a solid-phase synthesis of tetrakis(p-hydroxyphenyl)porphyrin-oligonucleotide hybrids. In these hybrids, up to four nucleic acid chains are linked to the phenolic substituents of the porphyrin via phosphodiester linkages. A representative hybrid with one oligonucleotide chain of the sequence TTAA was found to survive light irradiation under aerobic conditions for 2 h with less than 35% oxidation of the DNA chain. An assay measuring the diastereo- and enantioselectivity of the photosensitized ene reaction of mesitylol with singlet oxygen was set up that provides diastereomeric ratios via NMR of aliquots of the reaction solution. Enantiomers were separated gas chromatographically on a chiral stationary phase and were assigned based on the product distribution obtained with an enantiomerically enriched starting material. Our results are a starting point for the exploration of nanostructured reaction media based on DNA and porphyrins.

Easy introduction of maleimides at different positions of oligonucleotide chains for conjugation purposes

[2,5-Dimethylfuran]-protected maleimides were placed at both internal positions and the 3'-end of oligonucleotides making use of solid-phase synthesis procedures. A new phosphoramidite derivative and a new solid support incorporating the protected maleimide moiety were prepared for this purpose. In all cases maleimide deprotection (retro-Diels-Alder reaction) followed by reaction with thiol-containing compounds afforded the target conjugate.

Frequency of HLA‐A, ‐B and ‐DRB1 specificities and haplotypic associations in the population of Castilla y León (northwest‐central Spain)

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y León and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y León is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.

Maleimide-Dimethylfuran exo Adducts: Effective Maleimide Protection in the Synthesis of Oligonucleotide Conjugates

The reaction of maleimide-containing compounds with 2,5-dimethylfuran gives a mixture of exo and endo isomers from which the exo cycloadduct can be easily isolated taking advantage of its stability in concentrated aqueous ammonia. Bifunctional compounds incorporating a dimethylfuran-protected maleimide (exo adduct) have been attached to resin-linked oligonucleotide chains. Removal of protecting groups masking oligonucleotide functionalities followed by retro-Diels-Alder maleimide deprotection affords maleimido-oligonucleotides suitable for conjugation, as assessed by their reaction with different thiols.

Predicting DNA-mediated drug delivery in interior carcinoma using electromagnetically excited nanoparticles

Tumor-site-specific delivery of anti-cancer drugs remains one of the most prevailing problems in cancer treatment. While conventional means of chemo-delivery invariably leave different degrees of side-effects on healthy tissues, in recent times, intelligent chemical designs have been exploited to reduce the cross-consequences. In particular, the strategies involving superparamaganetic nanoparticles with surface assembled oligonucleotides as therapeutic carrier have raised affirmative promises. Process is designed in such a way that the therapeutic molecules are released preferentially at target site as the complementary oligonucleotide chains dissociate over the heat generated by the nanoparticles under the excitation of low frequency electromagnetic energy. In spite of the preliminary demonstrations, analytical comprehension of the entire process especially on the purview of non-trivial interactions between stochastic phase-transition phenomena of oligonucleotide chains and hierarchical organization of in vivo transport processes remains unknown. Here, we propose an integrated computational predictive model to interpret the efficacy of drug delivery in the aforementioned process. The basic physics of heat generation by superparamagnetic nanoparticles in presence of external electromagnetic field has been coupled with transient biological heat transfer model and the statistical mechanics based oligonucleotide denaturation dynamics. Conjunctionally, we have introduced a set of hierarchically appropriate transport processes to mimic the in vivo drug delivery system. The subsequent interstitial diffusion and convection of the various species involved in the process over time was simulated assuming a porous media model of the carcinoma. As a result, the model predictions exhibit excellent congruence with available experimental results. To delineate a broader spectrum of a priori speculations, we have investigated the effects of different tunable parameters such as magnetizing field strength, nanoparticle size, diffusion coefficients, porous media parameters and different oligonucleotide sequences on temperature rise and site-specific drug release. The proposed model, thus, provides a generic framework for the betterment of nanoparticle mediated drug delivery, which is expected to impart significant impact on cancer therapy.

Synthesis of Aminoglycoside-3′-Conjugates of 2′-O-Methyl Oligoribonucleotides and Their Invasion to a 19F labeled HIV-1 TAR Model

The potential of aminoglycosides to induce RNA-invasion has been demonstrated. For this purpose, aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides have been synthesized entirely on a solid phase. The synthesis includes an automated oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a (19)F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by (19)F NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 2'-O-methyl oligoribonucleotide (5'-CAGGCUCA-3'), has been obtained by the neomycin conjugate. The increased affinity results from a cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first molar equivalent of the conjugate induces hybridization of the second.

New acid photogenerators based on thioxanthen-9-one sulfonium derivatives for detritylation in the oligonucleotide synthesis

Photochemical activity of a number of cationic thioxanthen-9-one derivatives for the formation of the trityl cation was studied, which resulted in the selection of compounds suitable for photodetritylation. 10-(4-Heptyloxyphenyl)-9-oxo-2-(N,N,N-triethylammonio)methyl-9H-thioxanthenium bis(hexafluorophosphate) and 2-methyl-, 2-(2-methyl-1-propanoyl-2-tosyl)-, 1-chloro-4-propoxy-, and 2,4-diethyl-10-(4-heptyloxyphenyl)-9-oxo-9H-thioxanthenium hexafluorophosphates were found to be photoactivators of detritylation of 5′-O-(4,4′-dimethoxytrityl)thymidine. The detritylation reaction is the most efficient in dichloromethane. 2,4-Diethyl-10-(4-heptyloxyphenyl)-9-oxo-9H-thioxanthenium hexafluorophosphate was used as a detritylation photoactivator in the oligonucleotide synthesis using an automated DNA synthesizer. The yield in the elongation step of the oligonucleotide chain was 98%.

A mechanistic study of electron transfer from the distal termini of electrode-bound, single-stranded DNAs.

Electrode-bound, redox-reporter-modified oligonucleotides play roles in the functioning of a number of electrochemical biosensors, and thus the question of electron transfer through or from such molecules has proven of significant interest. In response, we have experimentally characterized the rate with which electrons are transferred between a methylene blue moiety on the distal end of a short, single-stranded polythymine DNA to a monolayer-coated gold electrode to which the other end of the DNA is site-specifically attached. We find that this rate scales with oligonucleotide length to the -1.16 ± 0.09 power. This weak, approximately inverse length dependence differs dramatically from the much stronger dependencies observed for the rates of end-to-end collisions in single-stranded DNA and through-oligonucleotide electron hopping. It instead coincides with the expected length dependence of a reaction-limited process in which the overall rate is proportional to the equilibrium probability that the end of the oligonucleotide chain approaches the surface. Studies of the ionic strength and viscosity dependencies of electron transfer further support this "chain-flexibility" mechanism, and studies of the electron transfer rate of methylene blue attached to the hexanethiol monolayer suggest that heterogeneous electron transfer through the monolayer is rate limiting. Thus, under the circumstances we have employed, the flexibility (i.e., the equilibrium statistical properties) of the oligonucleotide chain defines the rate with which an attached redox reporter transfers electrons to an underlying electrode, an observation that may be of utility in the design of new biosensor architectures.

Surface Effects in Water-Soluble Shell−Core Hybrid Gold Nanoparticles in Oligonucleotide Single Strand Recognition for Sequence-Specific Bioactivation

Highly monodisperse water-soluble shell-core hybrid gold nanoparticles were prepared by in situ reduction of gold salts in the presence of a bifunctional thiol, tiopronin. The resulting particles exhibited diameters of 20-60 nm, as characterized by scanning electron microscopy. The tiopronin coating was uniform, exposing a carboxylated monolayer surface that was further modified by coupling with an amino-maleimide linker. The polar heteroorganic shell rendered these materials water-soluble and, as a result, amenable to conditions for coupling with molecules of biological importance, for example, thiolated oligonucleotides. When brought into contact with a homogeneously dispersed oligonucleotide chain, the functionalized shell-core particle binds with a complementary oligonucleotide chain with high specificity. Binding could be qualitatively recognized by easily observed fluorescence differences. The largest particles (ca. 60 nm diameter) were unstable in buffered water, and further condensation ultimately led to aggregation and precipitation. In contrast, particles with diameters of 20-30 nm were stable in buffered water and were easily further functionalized with matched oligonucleotide double strands. This work thus constitutes a new route for the preparation of stable modified gold nanoparticles that can be easily further modified to deliver a metal core particle in aqueous media, as required for recognition, and manipulation, of specific biological sequences. Surface properties are key variables for these applications.

Synthesis of Oligonucleotide Glycoconjugates Using Sequential Click and Oximation Ligations

Oligodeoxyribonucleotide glycoconjugates bearing two trivalent glycoclusters have been synthesized by two alternative methods based on solid-supported oximation of aminooxy functionalized oligonucleotides with glycoclusters constructed by click chemistry. In more detail, the trivalent glycoclusters (5 and 6) bearing three sugar pendants were first assembled by treating a 4-[tri-O-propargylpentaerythrityloxy]benzaldehyde scaffold with methyl 6-azido-6-deoxyglycopyranoside under the click reaction conditions. Two phosphoramidite reagents containing a phthaloyl protected aminooxy function, viz., 2-cyanoethyl N,N-diisopropylphosphoramidites derived from 3-[3,5-bis(phthalimidoxymethyl)phenoxy]propanol (12) and 5-(4,4'-dimethoxytrityl)-1,2-dideoxy-1-C-(2-phthalimidoxyethyl)-beta-d-erythro-pentofuranose (16), were synthesized and incorporated as branching units in appropriate places of the oligonucleotide chains. On using 12, the phthaloyl protections of the branching unit were removed and two identical glycoclusters were attached via oxime linkage to the 5'-terminus of the support-bound oligonucleotide chain. With branching unit 16, the phosphoramidite coupling and the oximation were carried out alternately, allowing introduction of two dissimilar trivalent glycoclusters close to the 3'-end of the oligonucleotide chain. The products (20, 26) were released and deprotected by ammonolysis and purified by HPLC chromatography.

Preparation of short interfering RNA containing the modified nucleosides 2-thiouridine, pseudouridine, or dihydrouridine.

Modified uridine derivatives such as 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D) are naturally existing nucleoside units identified in tRNA molecules. Recently, we have shown that such base-modified units introduced into functionally important sites of siRNA modulate thermodynamic stability of the duplex and its gene silencing activity. In this unit, we describe chemical synthesis of 3'-phosphoramidite derivatives of s(2)U and D units (the 3'-phosphoramidite derivative of Psi is commercially available), and their use for the synthesis of RNA oligonucleotides according to the routine phosphoramidite protocol. The only exception concerns the oxidation step with I(2)/pyridine/water which, if applied towards oligonucleotides containing s(2)U units, would lead to the loss of sulfur. Therefore, to avoid this side reaction, tert-butyl hydroperoxide is used as an oxidizing reagent. After the oligonucleotide chain assembly is completed, the resulting oligomer is deprotected under mild basic conditions (MeNH(2)/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side-reaction during the routine synthesis of dihydrouridine-containing RNA. Oligonucleotides modified with s(2)U, D, or Psi units are useful models for structure-function studies. Here, the procedure for preparation of siRNA duplexes is described.