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Accueil > Publications > Recherche par années > Années 2000 > 2000

2000

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Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5’-protected thiol function and a 3 ’-amino group

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5’-ends and an amino group at their 3’-ends in good yield (up to 72 OD units/mu mol for a 19mer phosphorothioate). Syntheses of 3’-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2’-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol.

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Parallel-stranded DNA with Hoogsteen base pairing stabilized by a trans-[Pt(NH3)(2)](2+) cross-link : characterization and conversion into a homodimer and a triplex

The oligonucleotides 5’-d(TTTTCTTTTG) and 5’-d(AAAAGAAAAG) were cross-linked with a trans-[Pt(NH3)(2)](2+) entity via the N7 positions of the 3’-end guanine bases to give parallel-stranded (ps) DNA. At pH 4.2, CD and NMR spectroscopy indicate the presence of Hoogsteen base pairing. In addition, temperature-dependent UV spectroscopy shows an increase in melting temperature for the platinated duplex (35 degreesC) as compared to the non-platinated, antiparallel-stranded duplex formed from the same oligonucleotides (21 degreesC). A monomer-dimer equilibrium for the platinated 20mer is revealed by gel electrophoresis. At pH 4.2, addition of a third strand of composition 5’-d(AGCTTTTCTTTTAG) to the ps duplex leads to the formation of a triple helix with two distinct melting points at 38 degreesC (platinum crosslinked Hoogsteen part) and 21 degreesC (Watson-Crick part), respectively.

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Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors : Synthesis, biological properties, and antinociceptive activities

The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search for new analgesics devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general formula H3N+-CH(R-1)-P(O)(OH)-CH2-CH(R-2)-CONH-CH(R-3)-COO-, able to act as transition-state analogues and to fit the S-1, S-1’, and S-2’ subsites of both enzymes were designed. Selection of the R-1, R-2, and R-3 residues for optimal recognition of these enzymes led to the first dual competitive inhibitors with K-i values in the nanomolar range for neprilysin and aminopeptidase N. These compounds induce potent analgesic responses after intracerebroventricular or intravenous administrations in mice (hot plate test), and several of them were shown to be, at least, 10 times more potent than the previously described dual inhibitors.

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Polymeric substances and biofilms as biomarkers in terrestrial materials : Implications for extraterrestrial samples

Organic polymeric substances are a fundamental component of microbial biofilms. Microorganisms, especially bacteria, secrete extracellular polymeric substances (EPS) to form slime layers in which they reproduce. In the sedimentary environment, biofilms commonly contain the products of degraded bacteria as well as allochthonous and autochthonous mineral components. They are complex structures which serve as protection for the colonies of microorganisms living in them and also act as nutrient traps. Biofilms are almost ubiquitous wherever there is an interface and moisture (liquid/liquid, liquid/solid, liquid/gas, solid/gas). In sedimentary rocks they are commonly recognized as stromatolites.

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Projection methods for the analysis of complex motions in macromolecules

In studies of macromolecular dynamics it is often desirable to analyze complex motions in terms of a small number of coordinates. Only for simple types of motion, e.g., rigid-body motions, these coordinates can be easily constructed from the Cartesian atomic coordinates. This article presents an approach that is applicable to infinitesimal or approximately infinitesimal motions, e.g., Cartesian velocities, normal modes, or atomic fluctuations. The basic idea is to characterize the subspace of interesting motions by a set of (possibly linearly dependent) vectors describing elementary displacements, and then project the dynamics onto this subspace. Often the elementary displacements can be found by physical intuition. The restriction to small displacements facilitates the study of complicated coupled motions and permits the construction of collective-motion subspaces that do not correspond to any set of generalized coordinates. As an example for this technique, we analyze the low-frequency normal modes of proteins up to approximate to 20 THz (600 cm(-1)) in order to see what kinds of motions occupy which frequency range. This kind of analysis is useful for the interpretation of spectroscopic measurements on proteins, e.g., inelastic neutron scattering experiments.

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Psoralen adducts induced by triplex-forming oligonucleotides are refractory to repair in HeLa cells

The use of triple helix-forming oligonucleotides constitutes an attractive strategy to regulate gene expression by inhibition of transcription. Psoralen-oligonucleotide conjugates form, upon irradiation, covalent triplexes and thereby modify the specific target sequence. The processing of such photoproducts on the promoter of the gene coding for the interleukin-2 receptor a chain was investigated in HeLa cells and HeLa nuclear extracts. We demonstrate that psoralen cross-links are not repaired within the cell extracts nor inside cells. The mechanism of repair inhibition was elucidated in vitro : the presence of the third strand oligonucleotide inhibits the incision step of the damaged target by repair endonucleases. These results demonstrate the possibility of using this approach to induce a persistent intracellular DNA damage at a specific site and to afford prolonged transcription inhibition. (C) 2000 Academic Press.

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Radiolysis of nucleosome core DNA : a modelling approach

Purpose : To calculate the expected pattern of frank strand breaks (FSB) induced in the DNA of a nucleosome core particle (NCP) by the attack of radiolytic OH. radicals and to compare this with the experimental pattern of FSB induced by the in uitro irradiation of chicken NCP. Materials and methods : The structure of NCP was obtained from the PDB databank. Using molecular modelling, the structure of a linear DNA fragment with the central 60 bp sequence of NCP-DNA was determined. The accessibility of the sugar attack sites to OH. radicals at each nucleotide of the linear fragment or of the NCP-DNA was calculated. The probability of reaction of OH. with each sugar reactive sire was calculated using a Monte-Carlo method-based stochastic model.

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