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U Asseline

Publications and source records attributed to U Asseline.

At least 37 records · Page 2Linked to original sources
PubMed1990

Inhibition of simian virus 40 DNA replication in CV-1 cells by an oligodeoxynucleotide covalently linked to an intercalating agent.

An octathymidylate covalently linked via its 3'-end to an acridine derivative inhibited the cytopathic effect of Simian Virus SV40 on CV-1 cells in culture. Control experiments revealed that this effect was virus-specific and did not arise as a result of oligonucleotide degradation by nucleases. A photoactive probe was covalently attached to the 5'-end of the oligonucleotide-acridine conjugate. Upon UV-irradiation, photocrosslinking was shown to occur at the A. T-rich region within the viral origin of replication. A local triple helix can form at moderate salt concentrations with two octathymidylate-acridine conjugates bound to the octaadenylate sequence. Alternatively the octathymidylate-acridine conjugate can bind to the major groove of duplex DNA forming a local triple helix. Different mechanisms are discussed to explain the inhibition of viral DNA replication.

Acridines↗
PubMed1990

Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides.

Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.

Alkaloids↗
PubMed1989

Oligothymidylates covalently linked to an acridine derivative and with modified phosphodiester backbone: circular dichroism studies of their interactions with complementary sequences.

Oligothymidylates involving alternating alkyl phosphotriester-phosphodiester or methylphosphonate-phosphodiester backbones and covalently linked to an acridine derivative have been studied using circular dichroism. Two isomers with the same diastereoisomeric configuration for all the phosphotriesters (ethyl triester and neopentyl triester) or the methylphosphonate linkages were studied. These six compounds were compared to the parent oligonucleotide with unmodified phosphodiester bonds. Intramolecular interactions between the acridine and the bases of the oligonucleotides were revealed by the induced circular dichroism of the acridine dye. Binding to poly(rA) and poly(dA) induced large changes in the circular dichroism signal. All oligothymidylates formed double-stranded complexes with poly(rA). Substitution of phosphotriesters and methylphosphonates to phosphates allowed both double- and triple-stranded structures to be formed with with poly(dA). The double-stranded structures formed with poly(rA) and poly(dA) were characterized by different environments of the acridine dye. The circular dichroism spectra of the complexes with poly(dA) and the thermal stabilities of the complexes formed with both poly(rA) and poly(dA) were drastically dependent of the diastereoisomeric configuration of the phosphate modification. For the complexes formed with the pseudoequatorial stereoisomer the modification of the phosphate groups increased the stability of the complexes as compared with the oligothymidylate containing only phosphodiester linkages whereas it decreased it for pseudoaxial modifications.

Aminoacridines↗
PubMed1988

NMR studies of complex formation between the modified oligonucleotide d(T*TCTGT) covalently linked to an acridine derivative and its complementary sequence d(GCACAGAA).

The oligodeoxynucleotide d(TTCTGT) was covalently attached to the 9-amino group of 2-methoxy-6-chloro-9-aminoacridine (Acr) through its 3'-phOsphate via a pentamethylene linker (m5). In order to avoid its hydrolysis by nucleases inside the cel., one of its phosphates (TpT) was substituTed with a neopentyl group. Complex formation between each of the two purified isomers and the complementary strand d(GCACAGAA) was investigated by nuclear magnetic resonance. The COSY and NOESY connectivities allowed us to assign all the proton resonances of the bases, the sugars (except the overlapping 5'-5'' resonances), the acridine, and the pentamethylene chain. Structural information derived from the relative intensity of COSY and NOESY maps revealed that the duplex d(T*TCTGT).d(GCACAGAA) adopts a B-type conformation and that the deoxyriboses preferentially adopt a 2'-endo conformation. The NOE connectivities observed between the protons of the bases or the sugars and the protons of the dye show the intercalation of the acridine between the base pairs. NOE connectivities as well as imino proton resonances show that, at room temperature, the C7 base and the G8 base belonging to two different duplexes are paired. The pseudoaxial and pseudoequatorial isomers were assigned, and the differences in stability of their complex with the complementary strand are discussed.

Base Composition↗
PubMed1987

Oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent. Double helices with parallel strands are formed with complementary oligo-[beta]-deoxynucleotides.

An oligo-[alpha]-deoxynucleotide of sequence (5')d(TCTAAACTC) (3') was synthesized using the alpha-anomers of deoxynucleosides and its 5'-phosphate was covalently linked to a 9-amino acridine derivative via a pentamethylene linker. Two oligo-[beta]-deoxynucleotides containing the complementary sequence in either the 5'----3' or the 3'----5' orientation were synthesized using natural [beta]-deoxynucleosides. Complex formation was investigated by absorption and fluorescence spectroscopies. No change in spectroscopic properties was detected with the anti-parallel [beta] sequence. Absorption changes were induced in the visible absorption band of the acridine derivative at 2 degrees C when the acridine-substituted oligo-[alpha]-deoxynucleotide was mixed in equimolecular amounts with the complementary [beta]-sequence in the parallel orientation. Hypochromism was observed in the UV range. The fluorescence of the acridine derivative was quenched by the guanine base present in the second position of the complementary sequence. Cooperative dissociation curves were observed and identical values of melting temperatures were obtained by absorption and fluorescence. An increase in salt concentration stabilized the complex with a delta Tm of 8 degrees C when NaCl concentration increased from 0.1 to 1 M. These results demonstrate that an oligo-[alpha]-deoxynucleotide covalently linked to an intercalating agent is able to form a double helix with an oligo-[beta]-deoxynucleotide. The two strands of this [alpha]-[beta] double helix adopt a parallel 5'----3' orientation. The acridine ring is able to intercalate between the first two base pairs on the 5'-side of the duplex structure.

Base Sequence↗
PubMed1987

Oligo(alpha-deoxynucleotide)s covalently linked to intercalating agents: differential binding to ribo- and deoxyribopolynucleotides and stability towards nuclease digestion.

An octathymidylate was synthesized with the alpha anomer of thymidine instead of the naturally occurring beta anomer. This oligonucleotide binds to complementary sequences containing beta-nucleosides. Binding to ribose-containing oligomers and polymers is much stronger than binding to deoxyribose-containing analogs. A derivative of acridine (9-amino-6-chloro-2-methoxyacridine) was covalently attached either to the 5' phosphate or to the 3' phosphate of the alpha-octathymidylate. A pentamethylene linker was used to bridge the phosphate group and the 9-amino group of the acridine derivative. In both cases the complexes with the complementary sequences were strongly stabilized due to the additional binding energy provided by intercalation of the acridine ring within the miniduplex structure formed by the oligonucleotide with its target sequence. The acridine-substituted alpha-oligothymidylates did not lose their discrimination between ribose and deoxyribose-containing complementary sequences. The alpha-oligothymidylates were much more resistant towards endonucleases than their beta analogs, independently of whether they were linked to the acridine derivative. Acridine substitution provided additional protection against the corresponding exonucleases. alpha-Oligodeoxynucleotides covalently linked or not to intercalating agents represent families of molecules that open possibilities to block mRNA translation or viral RNA expression in vitro and in vivo.

Aminoacridines↗
PubMed1986

31P NMR studies of the binding of the oligonucleotide (Ap)3A to an oligodeoxythymidylate covalently linked to an acridine derivative.

31P NMR was used to study the specific interaction of an oligodeoxynucleotide containing four thymines and covalently attached to an acridine derivative through its 3'-phosphate [(Tp)4(CH2)5Acr] with a complementary oligoribonucleotide (Ap)3A. 31P-1H and 1H-1H chemical shift correlation spectroscopies were jointly used to provide the assignment of the phosphorus resonances. A downfield shift of two phosphorus resonances of (Tp)4(CH2)5Acr and of two phosphorus resonances of (Ap)3A was observed upon complex formation. The assignment of the phosphorus resonances which are downfield shifted allowed us to propose a model involving an equilibrium between several 1:1 complexes where the acridine ring is intercalated between different A.T base pairs.

Acridines↗
PubMed1985

Oligonucleotides covalently linked to intercalating agents. Influence of positively charged substituents on binding to complementary sequences.

A pentamethylene chain was used to covalently link the 3'-phosphate of oligothymidylates to the 9-amino group of an acridine derivative. Positively charged substituents were further attached to the 3'-phosphate group to form 3'-phosphotriesters. These molecules form specific complexes with poly(rA) which involve the formation of a number of A X T base pairs equal to that of thymines in the oligonucleotide. Absorption changes induced in the acridine absorption bands are similar to those expected upon intercalation of the acridine dye between A X T base pairs. The acridine covalently linked to the 3'-phosphate strongly stabilizes the complexes formed with poly(rA) as compared with the corresponding unsubstituted oligodeoxynucleotide. The presence of a positively charged substituent on the 3'-phosphate together with the acridine dye further enhances the interaction. The effect of salt concentration on complex stability depends on the number of negatively charged phosphate groups of the oligodeoxynucleotide and on the nature of the substituents borne by the 3'-phosphate group. When the oligothymidylate is substituted by an acridine dye, the stability of the poly(rA) complexes increases when salt concentration increases. If an additional positively charged substituent is present on the 3'-phosphate group, stability decreases when salt concentration increases for the shortest oligonucleotide (trimer) and increases with longer oligonucleotides. Thermodynamic parameters have been calculated from the concentration dependence of melting temperatures.

Base Sequence↗
PubMed1985

Proton and phosphorus nuclear magnetic resonance studies of an oligothymidylate covalently linked to an acridine derivative and of its binding to complementary sequences.

An oligodeoxynucleotide containing four thymines and covalently attached to an acridine derivative through its 3'-phosphate [(Tp)4(CH2)5Acr] was synthesized. Its conformation in solution was investigated by proton magnetic resonance. Both intramolecular interactions between the acridine dye and thymines and intermolecular interactions were demonstrated. Both proton and phosphorus magnetic resonances were used to study the specific interaction of (Tp)4(CH2)5Acr with poly(rA) and (Ap)3A. The results were compared to those obtained when the acridine-containing substituent was replaced by an ethyl group attached to the 3'-phosphate of the oligothymidylate. The acridine dye strongly stabilized the complexes formed with both poly(rA) and (Ap)3A. Upfield shifts of both adenine and acridine proton resonances were observed in the complexes. These results were ascribed to an intercalation of the acridine ring between A X T base pairs of the duplex structure formed by the oligothymidylate with its complementary oligoadenylate sequence. An analysis of proton and phosphorus chemical shifts as well as measurements of T1 relaxation times at different temperatures allowed us to propose several structures for the complexes formed by (Tp)4(CH2)5Acr with its complementary sequence.

Adenine Nucleotides↗
PubMed1984

Oligodeoxynucleotides covalently linked to intercalating dyes as base sequence-specific ligands. Influence of dye attachment site.

New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an intercalating dye. Visible absorption spectroscopy and fluorescence have been used to investigate the binding of poly(rA) to octadeoxythymidylates substituted by a 9-aminoacridine derivative in different positions along the oligonucleotide chain. The 9-amino group of the acridine dye was linked through a polymethylene bridge to the 3'-phosphate, the 5'-phosphate, the fourth internucleotidic phosphate or to both the 3'- and 5'-phosphates. Different interactions of the acridine dye were exhibited by these different substituted oligodeoxynucleotides when they bind to poly(rA). The interaction was shown to be specific for adenine-containing polynucleotides. The stability of these complexes was compared with that of oligodeoxynucleotides substituted by an alkyl group on the 3'-phosphate. The increase in stability due to the presence of the intercalating dye has been determined from the comparison of melting temperatures. These results are discussed with respect to the strategy of synthesis of a new class of molecules with high affinity and high specificity for nucleic acid base sequences.

Aminacrine↗
PubMed1984

Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides.

Oligodeoxyribonucleotides covalently linked to an intercalating agent via a polymethylene linker were synthesized. Oligothymidylates attached to an acridine dye (Acr) through the 3'-phosphate group [(Tp)n(CH2) mAcr ] specifically interact with the complementary sequence. The interaction is strongly stabilized by the intercalating agent. By using absorption and fluorescence spectroscopies, it is shown that complex formation between (Tp)n(CH2) mAcr and poly(rA) involves the formation of n A X T base pairs, where n is the number of thymines in the oligonucleotide. The acridine ring intercalates between A X T base pairs. Fluorescence excitation spectra reveal the existence of two environments for the acridine ring, whose relative contributions depend on the linker length (m). The binding of (Tp)4(CH2) mAcr to poly(rA) is analyzed in terms of site binding and cooperative interactions between oligonucleotides along the polynucleotide lattice. Thermodynamic parameters show that the covalent attachment of the acridine ring strongly stabilizes the binding of the oligonucleotide to its complementary sequence. The stabilization depends on the linker length; the compound with m = 5 gives a more stable complex than that with m = 3. These results open the way to the synthesis of a family of molecules exhibiting both high-affinity and high-specificity for a nucleic acid base sequence.

Acridines↗
PubMed1983

[New substances with high specific activity for nucleic acid sequences: oligodeoxynucleotides covalently linked to an intercalating agent].

New molecules are described in which an intercalating agent is covalently linked through a polymethylene chain to an oligodeoxynucleotide. Using an oligodT sequence covalently linked to an acridine dye, it is shown that these molecules bind in a specific way to their complementary sequence (poly (A] and that the stability is strongly enhanced by the intercalating agent as compared with the unmodified oligodT. Substitution of a phosphate group by a positively charged group strengthens complex formation and protects the neighboring phosphodiester bond against nuclease cleavage.

Intercalating Agents↗
PubMedDate not supplied

Chemical synthesis of natural and modified oligodeoxynucleotides.

This paper reports chemical synthesis of natural and modified oligodeoxynucleotides. In the first part after a short review of protective groups for the deoxynucleosides and for the phosphate groups, the different phosphorylating and coupling methods are listed and also the application fields for the different techniques (phosphotriester method in solution or solid-phase synthesis). The second part involves oligodeoxynucleotides in which phosphodiester groups are partially or fully replaced by alkylphosphotriester, phosphonate, phosphoramidate, phosphorothioate or phosphorothiolate groups. The last part describes synthesis of oligodeoxynucleotides bearing chemically reactive groups or an intercalating agent.

Base Sequence↗
PubMedDate not supplied

Oligodeoxynucleotides covalently linked to intercalating agents: a new class of gene regulatory substances.

Oligodeoxynucleotides have been covalently linked to a 9-aminoacridine derivative via their 3'-phosphate group. Specific complexes are formed with the complementary sequence of the oligonucleotide. The stability is strongly increased due to intercalation of the acridine derivative. Absorption, fluorescence, nuclear magnetic resonance and circular dichroism have been used to characterize complex formation. The stability of the complexes depends on the length of the linker between the acridine derivative and the 3'-phosphate group of the oligonucleotide. Oligonucleotides covalently linked to an intercalating agent can be used to selectively control gene expression. Transcription initiation can be blocked when such an oligonucleotide binds to the transcribed strand in the open complex formed by E. coli RNA polymerase with the bla promoter. With some oligonucleotides, non-specific effects on transcription can be detected, most probably due to binding of the modified oligonucleotide to RNA polymerase. Translation of the messenger RNA from gene 32 of phage T4 can be prevented by using an oligonucleotide complementary to the sequence upstream from the Shine-Dalgarno sequence. Inhibition of translation does not occur in the absence of the intercalating agent covalently linked to the oligonucleotide nor with oligonucleotides which do not have a target sequence on the mRNA.

DNA-Directed RNA Polymerases↗
PubMedDate not supplied

A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA.

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.

Animals↗
PubMedDate not supplied

Oligo-alpha-thymidylates covalently linked to intercalating agents: circular dichroism studies of their interaction with complementary sequences.

The interaction between oligo-alpha-thymidylates covalently linked to an intercalating agent (an acridine derivative) and their complementary sequences containing beta-nucleosides (poly(rA), poly(dA), r(Ap)7rA, p(dA)8) has been studied using circular dichroism spectroscopy. Binding to poly(rA) and to poly(dA) of the modified oligonucleotides led to large changes in the induced circular dichroism signal of the acridine ring. These changes depend on whether the dye is linked to the 3'- or to the 5'-end of the oligonucleotide. Interaction with poly(rA) as well as interaction with an octadeoxyriboadenylate led to the formation of a 1A:1T complex. With poly(dA), in addition to the 1A:1T complex, a 1A:2T complex was observed when the acridine derivative was linked to the 3'-end of the octa-alpha-thymidylate. The double-stranded structures formed with poly(rA) and poly(dA) were characterized by different environments of the acridine dye. Binding to poly(rA) gave much stronger complexes than binding to poly(dA). With poly(rA) the complex was more stable when the dye was bound at the 5'-end of the oligonucleotide. Comparison between the circular dichroism changes observed upon binding at the level of polymers [poly(rA) or poly(dA)] and those obtained at the level of oligomers [r(Ap)7rA or pd(A)8] gave information relative to the position of the acridine ring in the helix.

Aminoacridines↗
PubMedDate not supplied

Recognition of alternating oligopurine/oligopyrimidine tracts of DNA by oligonucleotides with base-to-base linkages.

A new concept is presented to design and synthesize modified oligonucleotides in order to extend the range of double-helical DNA sequences that can be recognized by oligonucleotides via triple helix formation. The DNA target is composed of adjacent oligopurine.oligopyrimidine domains where the oligopurine sequences alternate on the two DNA strands. Canonical (C,T)-motif triple helices are formed with each oligopurine.oligopyrimidine domain of the target sequence. The two third-strand oligonucleotides were joined together via an appropriate linker between the two terminal bases with either a 3'-3' or a 5'-5' polarity. Molecular modeling was used to predict the optimal length of the linker bridging two terminal bases. The interaction of DNA with such a modified oligonucleotide containing a C3'-3'U linkage was studied by thermal dissociation, footprinting, and gel retardation experiments. They provide experimental evidence that the oligonucleotide does form a switched triple helix on this extended DNA target sequence. The binding of the so-called "switch oligonucleotide" is enhanced as compared to the two unlinked parental oligonucleotides which form triple helices with each oligopurine.oligopyrimidine domain of the target sequence.

Base Sequence↗