Microwave heating for the rapid generation of glycosylhydrazides.

Conditions for simple derivatization of reducing carbohydrates via adipic acid dihydrazide microwave-assisted condensation are described. We demonstrate with a diverse set of oligo- and polysaccharides how to improve a restrictive and labor intensive conventional conjugation protocol by using microwave-assisted chemistry. We show that 5 min of microwave heating in basic or acidic conditions are adequate to generate, in increased yields, intact and functional glycosylhydrazides, whereas hours to days and acidic conditions are generally required under conventional methods.

New acridone derivatives for the electrochemical DNA-hybridisation labelling.

In the field of DNA sensing, DNA hybridisation detection is generally performed by fluorescence microscopy. However, fluorescence instrumentation is difficult to miniaturise in order to produce fully integrated DNA chips. In this context, electrochemical detection of DNA hybridisation may avoid this limitation. Therefore, the use of DNA intercalators is particularly attractive due to their selectivity toward DNA double strand enabling DNA labelling without target chemical modification and, for most of them, to their electroactivity. We have synthesized a pyridoacridone derivative dedicated to DNA hybridisation electrochemical-sensing which presents good electrochemical reversibility, electroactivity at mild potentials and specificity toward DNA double strand. The electrochemical behaviour of this molecule has been assessed using cyclic voltammetry (CV). DNA/intercalator interactions were studied by differential pulse voltammetry (DPV) before application to hybridisation detection onto DNA sensors based on polypyrrole modified electrodes.

Electropolymerisable pyrrole-oligonucleotide: synthesis and analysis of ODN hybridisation by fluorescence and QCM.

Conducting polymer films, such as polypyrrole, appear particularly attractive for the immobilisation of biological molecules by entrapment or covalent grafting. We describe here a new pyrrole phosphorarnidite building block allowing the synthesis of oligonucleotide (ODN) bearing a pyrrole moiety. The electropolymerisable pyrrole moiety was then introduced on the 5' end of the oligonucleotide. The electrosynthesis of a copolymer, from solutions containing pyrrole and pyrrole-ODN, gives in one step strongly adhesive films containing ODN probes at electrode surfaces. In this contribution, we have used such a methodology to verify its feasibility for the modification of quartz crystal microbalance (QCM) electrodes. The obtained biosensors enable the detection of DNA hybridisation in real time by micro-gravimetric transduction. Finally, as DNA targets were previously modified by biotin, we have used the affinity between biotin and avidin to validate the effectiveness of QCM transduction by fluorescence microscopy and to amplify the recorded micro-gravimetric signal.

Electropolymerization as a versatile route for immobilizing biological species onto surfaces. Application to DNA biochips.

Biosensors based on electronic conducting polymers appear particularly well suited to the requirements of modern biological analysis--multi-parametric assays, high information density, and miniaturization. We describe a new methodology for the preparation of addressed DNA matrices. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety. The resulting polymer film deposited on the addressed electrode consists of pyrrole chains bearing covalently linked oligonucleotides (ODN). An oligonucleotide array was constructed on a silicon device bearing a matrix of 48 addressable 50 x 50 microns gold microelectrodes. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Fluorescence detection results show good sensitivity and a high degree of spatial resolution. In addition, gravimetric studies carried out by the quartz crystal microbalance technique provide quantitative data on the amount of surface-immobilized species. In the case of ODN, it allows discrimination between hybridization and nonspecific adsorption. The need for versatile processes for the immobilization of biological species on surfaces led us to extend our methodology. A biotinylated surface was obtained by coelectropolymerization of pyrrole and biotin-pyrrole monomers. The efficiency for recognition (and consequently immobilization) of R-phycoerythrin-avidin was demonstrated by fluorescence detection. Copolymerization of decreasing ratios of pyrrole-biotin over pyrrole allowed us to obtain a decreasing scale of fluorescence.

Characterization and optimization of a real-time, parallel, label-free, polypyrrole-based DNA sensor by surface plasmon resonance imaging.

We describe in this paper a methodology to quantify multispot parallel DNA hybridizations and denaturations on gold surfaces by using, on one hand, a polypyrrole-based surface functionalization based on an electrospotting process and, on the other hand, surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time. Two characterization steps were performed in order to optimize the immobilization of oligonucleotide probes and, thus, to increase the signal-to-noise ratio of monitored hybridization signals: the first step consisted of characterizing the signal dependence upon the density of immobilized 15-mer probes, and, the second step, in analyzing the hybridization response versus spot thickness. We further demonstrated that a surface density of polypyrrole/DNA probes of approximately 130 fmol/ mm2 (590 pg/mm2) optimizes the hybridization signal that can be detected directly. Optimal thickness of the spot was found to be close to 11 nm. Specificity and regeneration steps on each spot have also been demonstrated successfully, showing this method to be very competitive and convenient in use.

Electroconducting polymers for the construction of DNA or peptide arrays on silicon chips.

We wish to show in this paper new developments and new applications of the pyrrole copolymerization process allowing the addressing of pyrrole-modified biomolecules on microelectrode arrays. Two main developments are described: the first one concerns the development of multiplexed silicon chips bearing 128 microelectrodes instead of 48 for the passive chips. The second one deals with new applications of this grafting process concerning not only DNA chips but peptide chips too. In this way, copolymerization of pyrrole peptides on the chip (leading to peptide chip) and their immunological detection is illustrated. This technology shows a high dimensional resolution and a real versatility.

Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping.

We describe in this article an oligonucleotide array constructed on a silicon device bearing a matrix of addressable 50-microns microelectrodes. Each electrode was covered by a conducting polymer (polypyrrole) grafted by an oligonucleotide (ODN). The DNA chip was prepared by successive electrochemically addressed copolymerizations of 5' pyrrole-labeled ODN and pyrrole. Following hybridization of the biotinylated amplified sample on the chip bearing a series of probes, detection was carried out by fluorescence microscopy through an R-phycoerythrin label. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Results show good sensitivity and a high degree of dimensional resolution.

Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group.

A new methodology for the preparation of addressed DNA matrices is described. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5' end a pyrrole moiety introduced by phosphoramidite chemistry. The electro-controlled synthesis of the copolymer (poly-pyrrole) gives, in one step, a solid conducting film deposited on the surface of an electrode. The resulting polymer consists of pyrrole chains bearing covalently linked oligonucleotide. The polymer growth is limited to the electrode surface, so that it is possible to prepare a DNA matrix on a multiple electrode device by successive copolymerizations. A support bearing four oligonucleotides was used to detect three ras mutations on a synthetic DNA fragment.

Chemical synthesis of a biologically active natural tRNA with its minor bases.

The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

[Total chemical synthesis of natural transfer RNA]. / Synthèse chimique totale d'un ARN de transfert naturel.

New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is used to detect Human Papillomavirus types 16 and 18 in cells and biopsies and for the specific colorimetric detection of HIV1 in extracted DNA.

Topography and ontogeny of the neurons expressing vasopressin, oxytocin, and somatostatin genes in the rat brain: an analysis using radioactive and biotinylated oligonucleotides.

1. The use of radioactive and biotinylated oligonucleotide probes has been optimized to detect and analyze by in situ hybridization, neurons expressing neuropeptide genes (vasopressin, oxytocin, somatostatin). 2. In situ hybridization was performed on cryostat-cut sections obtained from tissues perfused with 1% formaldehyde. Radioactive probes were labeled by tailing with 35S-dATP and revealed with autoradiography. Biotinylated probes were obtained either by the incorporation of 11-biotin dUTP or by the addition of biotinylated nucleotides to the oligonucleotide during its synthesis. Biotin was revealed with streptavidin alkaline phosphatase and the appropriate substrate. 3. In the adult rat brain, radioactive and biotinylated probes revealed peptidergic neurons. The biotinylated probes provided an optimal cellular and subcellular resolution with a sensitivity similar to that observed with radioactive probes. Staining was selectively restricted to the cytoplasm and to the proximal part of processes. 4. Biotinylated vasopressin probes with 10 biotins added demonstrated magnocellular neurons and parvocellular neurons in the suprachiasmatic nucleus and the bed nucleus stria terminalis. 5. Vasopressin gene expression was studied during ontogeny in the rat fetus and neonate. Vasopressin mRNA was first detectable at gestational day 16 in the supraoptic nucleus in neurons of neuroblastic appearance. An aspect similar to the one present in adult was found at gestational day 19 in magnocellular neurons and at day 3 postnatal in parvocellular neurons. 6. The results confirm that radioactive oligonucleotide probes are efficient tools to investigate neuropeptide gene expression by in situ hybridization and demonstrate that biotinylated oligonucleotides are very efficient and provide a much higher resolution than radioactive probes with a reasonable sensitivity.

Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl.

The synthesis of protected nucleoside phosphoramidites bearing various markers such as biotinyl, dinitrophenyl, dansyl and pyrenyl groups are reported. These labelled deoxynucleosides phosphoramidites were used for solid phase oligonucleotide synthesis in the same way than the usual protected phosphoramidities without any change in the synthetic cycle and the deprotection step. The new labelled building blocks described herein have been used in conjunction with the labile base protected phosphoramidites ('PAC phosphoramidites') which allowed mild ammonia deprotection, especially recommended for the dinitrophenyl-labelled oligonucleotides. Multiple labelling (i.e. 10 to 20 biotins) can be efficiently and easily performed, on the same oligonucleotide which results in an increase of sensitivity. The polylabelled oligonucleotides are chemically well defined and gave increased signal and low background coloration for in situ hybridisation. The modified oligonucleotides can still be kinased in the normal way as the reporter groups are on the heterocycles.

Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence.

A single-point mutation, consisting of a T-to-G transversion, was made in the third nucleotide of the conalbumin gene T-A-T-A-A-A-A homology sequence (the T-A-T-A or Goldberg-Hogness box). In an in vitro system, specific transcription of the mutant DNA was drastically decreased compared to the normal gene. This down-mutation is consistent with the idea that the T-A-T-A box is an important element for specific initiation of transcription.

A novel class of condensing reagents in phosphodiester oligodeoxyribonucleotides synthesis. Application of the constituents of free terminal carboxy oxytocine gene.

Benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium salts have been used as novel condensing agents to promote internucleotide bond formation in phosphotriester oligodeoxyribonucleotide synthesis. The effectiveness of these stable compounds has been shown by the synthesis of the six oligodeoxyribonucleotide building blocks of the carboxy terminal oxytocine gene. Some modifications incorporated in the phosphotriester method include a rapid procedure to prepare the fully protected dideoxyribonucleotide blocks and size exclusion chromatography of the deprotected oligodeoxyribonucleotides. The triester oligodeoxyribonucleotides were studied by chemical ionization mass spectrometry.