Biomedical subjects

U Asseline

Publications and source records attributed to U Asseline.

At least 19 recordsLinked to original sources

PubMed2001

Synthesis and properties of triple helix-forming oligodeoxyribonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine.

Multiple incorporations of 7-chloro-7-deaza-2'-deoxyguanosine in place of 2'-deoxyguanosine have been performed into a triple helix-forming oligodeoxyribonucleotide involving a run of six contiguous guanines designed to bind in a parallel orientation relative to the purine strand of the DNA target. The ability of these modified oligodeoxyribonucleotides to form triple helices in a buffer containing monovalent cations was studied by UV--melting curves analysis, gel shift assay and restriction enzyme protection assay. In the presence of Na(+), the incorporation of two, three or five modified nucleosides in the third strand has improved the efficacy of formation of the triplex as compared to that formed with the unmodified oligonucleotide. The stabilities of the three modified triplexes were similar. The coupling of 6-chloro-2-methoxy-9-(omega-hexylamino)-acridine to the 5'-end of the oligonucleotides containing modified nucleosides led to an increase in triplex stability similar to that observed when the acridine was added to the 5'-end of the unmodified oligonucleotide. In the presence of K(+), only the oligodeoxyribonucleotides containing modified G retained the ability to form triple helices with the same efficiency. The incorporation of the modified nucleoside has two effects: (i) it decreases TFO self-association, and (ii) it slightly increases triplex stability. The enhanced ability of the modified oligonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine over the parent oligomer to form triple helices was confirmed by inhibition of restriction enzyme cleavage using a circular plasmid containing the target sequence.

DNA, Viral↗

PubMed2001

Oligonucleotide-conjugated thiazole orange probes as "light-up" probes for messenger ribonucleic acid molecules in living cells.

"Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

Absorptiometry, Photon↗

PubMed2000

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 alpha 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.

Base Sequence↗

PubMed2000

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 phosphor-othioate 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/micromol 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 phosphor-amidite 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. This proced-ure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S -acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3'-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.

Amides↗

PubMed2000

Linkage of a triple helix-forming oligonucleotide to amsacrine-4-carboxamide derivatives modulates the sequence-selectivity of topoisomerase II-mediated DNA cleavage.

Amsacrine-4-carboxamide-oligonucleotide conjugates were synthesized and studied for their capacity to form DNA triple helices and to alter human topoisomerase II binding and cleavage properties. The intercalating agent was attached to the 3'- or the 5'-end of a 24 nt triple helix-forming oligonucleotide via linkers of different lengths. The stability of these DNA triple helices was investigated by gel retardation and melting temperature studies using a synthetic 70 bp DNA duplex target. The effect of the conjugates on DNA cleavage by topoisomerase II was evaluated using the 70 bp duplex and a 311 bp restriction fragment containing the same triple helix site. The conjugate with the amsacrine derivative linked to the 3' end of the TFO via a hexaethylene glycol linker modulates the extent of DNA cleavage by topoisomerase II at specific sites.

Amsacrine↗

PubMed1999

Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents.

The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA+along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilizedtemperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.

Alkynes↗

PubMed1999

Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways.

Psoralen is an asymmetric photoreactive intercalator with a furane and a pyrone side. When intercalated at 5'-TpA-3' sites and upon UVA irradiation, the psoralen can react with the thymine residues on both strands, introducing an interstrand crosslink. Using psoralen-coupled triple-helix-forming oligonucleotides, psoralen interstrand crosslinks can be site-specifically introduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors. In addition, crosslinks introduced via a triple-helix-forming oligonuleotide are oriented with the furane side of the psoralen associated with a specific strand of the target sequence. Here, the transformation efficiency, the mutation frequency and the mutational spectra of site-specifically placed and oriented crosslinks were examined in yeast cells. We found that the nature of the targeted mutations depended on the crosslink orientation: bypass of the pyrone-adducted thymine yielded T-->A or T-->C substitutions and A insertions, while bypass of the furane-adducted thymine yielded T-->G substitutions and G insertions. Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis. In addition, the observed pattern of mutagenesis suggests a coupling to transcription, similar to the one observed in mammalian cells. Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed strand, suggesting that the incision preference of psoralen crosslinks, which has been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells.

Base Sequence↗

PubMed1998

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

Adenine↗

PubMed1997

Modification of DNA duplexes to smooth their thermal stability independently of their base content for DNA sequencing by hybridization.

The possibility of equalizing DNA duplex stability is essential for the application of sequencing by hybridization. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their base content. Modified *C bases have been developed and incorporated into oligonucleotides. The influence of these modifications on duplex stability has been studied by absorption spectroscopy, thus allowing selection of N -4-ethyl-2'-deoxycytidine (d4EtC), which hybridizes specifically with natural dG to give a G4EtC base pair whose stability is very close to that of natural AT base pairs. Duplexes built with AT and/or G4EtC base pairs exhibit thermal stabilities independent of their base content in a classical buffer solution, thus enabling control of the stability of DNA hybrids as a function of their length only.

Base Composition↗

PubMed1997

Antisense properties of end-modified oligonucleotides targeted to Ha-ras oncogene.

Phosphodiester oligodeoxyribonucleotides linked to an intercalating agent or a dodecanol tail or both complementary to the 12th codon region of Ha-ras mRNA were compared with the unmodified oligonucleotides of the same size and sequence with respect to their ability to induce RNaseH cleavage and antisense activity in cell culture. The hydrophobic tail not only protected the oligonucleotide from nucleases but also enhanced RNase H cleavage of the target. Oligonucleotides carrying both an acridine and a dodecanol substituent inhibited the proliferation of HBL100ras1 cells (human mammary cells stably transformed with the T24 Ha-ras gene carrying a G-->T point mutation in codon 12) at a 20-fold to 30-fold lower concentration than unmodified ones. Therefore, these modified oligonucleotides may prove useful for antisense applications.

Base Sequence↗

PubMed1994

Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare the reactivity of the activatable dodecamers (cleavage of the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reactivity of the RNA with the dodecamers was weaker than that of the DNA target. With psoralen-substituted oligonucleotides, it was possible to obtain complete discrimination between the mutated target (which contained a psoralen-reactive T(U) in the 12th codon) and the normal target (which contained G at the same position). When longer Ha-ras RNA fragments were used as targets (120 and 820 nucleotides), very little reactivity was observed. Part of the reactivity could be recovered by using 'helper' oligonucleotides that hybridized to adjacent sites on the substrate. A 'helper' chain length greater than 13 was required to improve the reactivity of dodecamers. However, the dodecanucleotides induced RNase H cleavage of the target RNA in the absence of 'helper' oligonucleotide. Therefore, in the absence of the RNase H enzyme, long oligonucleotides are needed to compete with the secondary structures of the mRNA. In contrast, formation of a ternary complex oligonucleotide-mRNA-RNase H led to RNAT cleavage with shorter oligonucleotides.

Base Sequence↗

PubMed1993

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Base Sequence↗

PubMed1993

HIV gene regulatory proteins tat and rev and their interactions with synthetic RNA.

Synthetic oligoribonucleotides have been prepared and annealed to form model RNA duplexes that mimic the high affinity RNA recognition sites for the HIV-1 tat and rev proteins. The contributions of individual functional groups on the model RNAs to the specificity of binding by their respective proteins were studied by use of oligoribonucleotides containing site-specifically modified nucleotides. Both tat and rev appear to recognise specifically a limited number of functional groups in the major groove of an RNA double helix distorted by virtue of unpaired or non-Watson-Crick paired nucleotides.

Base Sequence↗

PubMed1992

A novel strategy for site-directed chemical reactions in single stranded DNA--absorption and NMR spectroscopic studies of model compounds.

A new and simple model enabling a chemical species to be brought to a preselected site in single strand DNA is reported. Two oligonucleotides containing a propanediol linkage were hybridized to their complementary sequences with an extra-base opposite the propanediol derivative. Absorption studies results shown that the addition of a bisacridine derivative strongly increased the stabilities of both duplexes when added in a 1:1 ratio. NMR studies on one of these duplexes brought evidence of the intercalation of the bisacridine at the position involving the propanediol linkage. These results suggest that this system could be used to target a specific reaction at a preselected position using the bisacridine derivative as carrier for the reactive species.

Acridines↗

PubMed1991

Synthesis and physicochemical properties of oligonucleotides built with either alpha-L or beta-L nucleotides units and covalently linked to an acridine derivative.

Modified deoxynucleosides 2'-deoxy-beta-L-uridine, beta-L-thymidine, alpha-L-thymidine, 2'-deoxy-beta-L-adenosine and 2'-deoxy-alpha-L-adenosine were synthesized and assembled as homooligomers, respectively: octa-beta-L-deoxyuridylates, octa beta-L and alpha-L-thymidylates and tetra beta-L and alpha-L-deoxyadenylates. These unnatural oligomers were then substituted with an acridine derivative. The binding studies of these modified oligonucleotides with D-ribo- and D-deoxyribopolynucleotides were carried out by absorption spectroscopy. While beta-L-d(Up)8m5Acr, beta-L-(Tp)8m5Acr, alpha-L-(Tp)8m5Acr did not interact with poly(rA) and poly(dA), beta-L-d(Ap)4m5Acr and alpha-L-d(Ap)4m5Acr did form double and triple helices with poly(rU) and poly(dT), respectively. Their stability towards nuclease digestion was studied through comparison with that of octa-beta-D-thymidylate and tetra beta-D-deoxyadenylate covalently linked to an acridine derivative. One endonuclease (nuclease P1 from Penicillium citrinum) and two exonucleases (a 3'-exonuclease from Crotalus durissus venom and a 5'-exonuclease extracted from calf thymus) were employed. beta-L- and alpha-L-oligomers demonstrate a high resistance toward nuclease digestion.

Acridines↗

PubMed1991

Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates.

Base-pair sequences in double-stranded DNA can be recognized by homopyrimidine oligonucleotides that bind to the major groove at homopurine.homopyrimidine sequences thereby forming a local triple helix. To make oligodeoxynucleotides resistant to nucleases, we replaced the natural (beta) anomers of the nucleotide units by the synthetic (alpha) anomers. The 11-mer alpha oligodeoxynucleotide 5'-d(TCTCCTCCTTT)-3' binds to the major groove of DNA in an antiparallel orientation with respect to the homopurine strand, whereas a beta oligonucleotide adopts a parallel orientation. When an intercalating agent was attached to the 3' end of the alpha oligodeoxynucleotide, a strong stabilization of the triple helix was observed. A 16-base-pair homopurine.homopyrimidine sequence of human immunodeficiency virus proviral DNA was chosen as a target for a 16-mer homopyrimidine alpha oligodeoxynucleotide. A restriction enzyme that cleaves DNA at the junction of the homopurine.homopyrimidine sequence was inhibited by triple-helix formation. The 16-mer alpha oligodeoxynucleotide substituted by an intercalating agent was approximately 20 times more efficient than the unsubstituted oligomer. Nuclease-resistant alpha oligodeoxynucleotides offer additional possibilities to control gene expression at the DNA level.

Base Sequence↗

PubMed1991

Recognition and photo-induced cleavage and cross-linking of nucleic acids by oligonucleotides covalently linked to ellipticine.

Oligopyrimidines covalently linked to ellipticine derivatives form duplex and triplex structures with target single-stranded oligopurine sequences. They also bind to duplex DNA at homopurine-homopyrimidine sequences where they form local triple helices. Irradiation at wavelengths longer than 300 nm of the complex formed by an oligonucleotide-ellipticine conjugate with its target sequence induced (i) cleavage of the target at bases located in close proximity to the dye and (ii) cross-linking of the target sequence to the derivatized oligonucleotide. Both cross-linking and cleavage reactions decreased when temperature increased with a half-transition corresponding to the dissociation of the oligonucleotide-ellipticine conjugate from its target nucleic acid, demonstrating that the observed photochemical effects are dependent on hybrid formation. When the target was a double-stranded DNA, photochemical reactions were observed on both strands of the duplex. Photo-induced cross-linking was more efficient than cleavage when the target was single-stranded; the reverse was observed when the target was duplex DNA.

Base Sequence↗

PubMed1991

Synthesis of site-specifically modified oligoribonucleotides for studies of the recognition of TAR RNA by HIV-1 tat protein and studies of hammerhead ribozymes.

Synthetic oligoribonucleotides having single uracil residues replaced by dU, dT, 2'-O-methylU or 5-bromodU have been prepared and used in the study of the interaction of HIV-1 tat protein with an RNA stem-loop. The preparation of phosphoramidites of 5-bromouridine and purine riboside suitable for use in solid-phase oligoribonucleotide synthesis is also described. The effect of adenine replacement by purine in a hammerhead ribozyme has also been determined.

Base Sequence↗