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Antisense oligonucleotides are promising medicines for treating various diseases, although their efficiency still requires high doses. Their delivery in the cytosol and nucleus to reach their mRNA targets would increase their efficiency at the same time as reducing the dose.
We conjugated the histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) at the 5’-end of the RNase H-incompetent antisense 2’-O-methyl-phosphodiester oligonucleotide (2’-Ome RNA705) targeting aberrant splicing of luciferase pre-mRNA in HeLa pLuc705 cells. H5WYG was also conjugated with 2’-Ome-RNA705 labelled by fluorescein at the 3’-end. Then, H5WYG-2’-Ome-RNA705 conjugate and 2’-Ome-RNA705 were formulated with lipofectamine to favor their uptake in HeLa pLuc705 cells.
Confocal microscopy showed that, after 4 h and overnight incubation, the presence of fluorescein-labelled 2’-Ome-RNA705 in the cytosol and nucleus was enhanced when the oligonucleotide was conjugated with H5WYG. We found that H5WYG-2’-Ome-RNA705 increased the splicing redirection and restoration of a functional luciferase mRNA. Luciferase activity and luciferase mRNA levels in these cells were 6.6- and two-fold higher, respectively, with H5WYG-2’-Ome-RNA705 than with 2’-Ome-RNA705.
The results of the present study show that the conjugation of 2’-Ome antisense RNA to peptide H5WYG is a good strategy for improving its cytosol delivery, accumulation in the nucleus and antisense activity.
The synthesis of a pyrazolo[1,5-a]-1,3,5-triazine C-nucleoside (dA(PT)), designed to form two hydrogen bonds with a complementary dT residue, is reported. Oligonucleotides including this dA nucleoside analogue possess base-pairing properties similar to those of the parent oligonucleotide. This dA nucleoside analogue is more resistant to acid-catalyzed hydrolysis than dA.
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
2’-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribonucleotides. Single and triple incorporations in various arrangements were performed. The Watson and Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves, CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with DNA-DNA and DNA-RNA duplexes involving dU at the same positions. Oligonucleotides containing Un clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with higher stabilities for the DNA-RNA duplexes similar to the one of the parent DNA-RNA duplex. Investigations into the thermodynamic properties of these 17-base-pair duplexes revealed [capital Delta]G values (37°C) that are in line with the measured Tm values for both the DNA-DNA and DNA-RNA duplexes. CD spectroscopic structural investigations indicated that the conformations of the DNA-DNA and DNA-RNA duplexes involving Un are similar to those of the dT-rA and dU-rA containing duplexes. Only small changes in intensities and weak blue shifts were observed when three Uns were incorporated into the duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of both the DNA-DNA and RNA-DNA duplexes. However, molecular dynamic simulations indicated that the Uns adopt a C2’-exo sugar pucker conformation close to an A-helix type without perturbing the C2’-endo sugar pucker conformations of their 2’-deoxynucleoside neighbours. These results confirm the potential of 2’-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic strategies.
We recently reported the design of new fluorescent oligo-2’-deoxyribonucleotides (FODNs) for the detection of terminal mismatches on DNA duplexes in homogeneous assays. We now report the validation of this method in homogeneous assays with other sequences and the feasibility of the detection of terminal mismatches with immobilized FODNs. In all cases studied, the mismatched duplexes were more fluorescent than the perfect ones and results confirmed that the discrimination factor is sequence-dependent.
This paper describes the design of terminal-mismatch discriminating fluorescent oligonucleotides (TMDFOs). The method is based on the use of sets of oligo-2’-deoxyribonucleotide probes linked via their 5’-ends, and varying-sized flexible polymethylene chains, to thiazole orange, with the linker being attached to the benzothiazole moiety. The sequence of each set of labelled probes was identical and complementary to the sequence to be analyzed on the single-stranded nucleic acid target except at the interrogation position, located at the 5’-end of the probes in a position adjacent to the attachment site of the label, where each of the four nucleic bases were incorporated. This work allowed the selection of probes showing, upon their hybridization with the target sequence, good discrimination between the matched and the mismatched duplexes under non-stringent conditions, with the mismatched duplexes being more fluorescent than the perfectly matched ones.
Because the influence of the chemical structure of monomethine cyanine-oligo-2’-deoxyribonucleotide (ODN) conjugates on their binding and fluorescence properties has remained largely undetermined, we synthesized and studied a wide range of conjugates with various structural patterns. Different cyanine dyes such as thiocyanine, quinocyanine, and thiazole orange isomers were obtained. In the case of unsymmetrical cyanines, the linker was attached to either the quinoline or the benzothiazole nucleus. The influence of the ODN counterpart was evaluated by linking the cyanines to the 5’-end or to an internucleotidic phosphate. In the first case, the influence of neighboring nucleic bases was studied, whereas in the second, the stereochemical configuration at the phosphorus atom bearing the cyanine was investigated. We report here on relationships between the structures of the dyes and conjugates and some of their properties, such as the stability and fluorescence changes observed on their hybridization with the target sequence. This study provides useful information towards the design of ODN-cyanine conjugates.
Fluorescent oligonucleotides (FONs) are used in a wide variety of areas such as molecular and mechanistics biological studies, molecular diagnostics, therapeutic development, biotechnology and nanotechnology. Ever since the post-genome era, there has been an ever-increasing demand for more rapid and accurate nucleic acid detection and quantification methods. Genetic information analyses require highly sensitive and specific detection of certain sequences, single nucleotide changes, specific structures and varied reactions in different formats in vitro, in living cells and, ultimately in animals and in human beings. Ideally, a unique event Could be detected and quantified using the genomic information without amplification of the nucleic acids to be analyzed. Recent developments enabling detection at the single-molecule (SM) level have opened new perspectives for applications.
Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences ; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities ; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5’-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate : by SPIR measurements at 37 degrees C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life similar to 10 h (50-fold longer compared with the unconjugated TFO/ LNA).
We report here the synthesis of oligo-2’-deoxyribonucleotides (ODNs) conjugated with perylene. Introduction of perylene, coupled either directly or via a propyl linker to the anomeric position of a 2’-deoxyribose residue, induces the formation of two anomers. Single incorporations of each pure anomer of these sugar perylene units have been performed at either the 5’-end or an internal position of a pyrimidic pentadecamer. The binding properties of these modified ODNs with their single- and double-stranded DNA targets were studied by absorption spectroscopy. Double incorporations of the sugar perylene unit most efficient at stabilizing the triplex and duplex structures (the beta-anomer involving the propyl linker) have been performed at both the 5’-end and at an internal position (or both the 5’- and 3’-ends) of the ODN chain.
We report here a novel, simple reagent enabling the chemical incorporation of a thiophospliate or a phosphate group at the 5’-end of oligonucleotides using very mild basic deprotection conditions. This method can be useful in the case of alkali sensitive modified oligonucleotides. This reagent also gives access to the preparation of bifunctional oligonucleotides with either a thiophosphate group at the 5’-end and a phosphate at the 3’-end, or two thiophosphate groups at both the 5’- and the 3’-ends, or a 5’-thiophosphate group and a 3’-amino-containing linker. (C) 2004 Elsevier Ltd. All rights reserved.
DNA binding compounds, such as benzo[e] (BePI) and benzo[g] pyridoindole (BgPI) derivatives, exhibit preferential stabilization of triple helices. We report here the synthesis of a series of pyrimidine triple-helix-forming oligo-2’-deoxyribonucleotides conjugated with these molecules. BePI was coupled to the 5-position of 2’-deoxyuridine via two linkers of different sizes attached to its 11-position and placed at either the 5’-end, inside the sequence, or at both the 5’-end and the internal positions using periodate oxidation of a diol-containing oligonucleotide followed by reductive coupling with amino-linked BePI. The same BePI derivatives were also linked to the oligonucleotide chain via internucleotidic phosphorothiolate or phosphoramidate linkages. A mixture of diastereoisomers was prepared as well as separate pure Rp and Sp isomers.
Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming oligo-2’-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-2’-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the parent phosphodiester sequences.
We report here the synthesis and binding properties of oligonucleotides involving a perylene unit linked to the anomeric position of a 2’-deoxyribose residue. Both anomers were separated and incorporated separately at either the 5’-end or the internal position of a pyrimidine sequence. In any case the presence of the perylene unit stabilizes the complexes formed with either the single or the double-stranded target.
The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophillic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2’-deoxyriboadenylate (dA(15)), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp) ; these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (Phi(fl)) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5’[CACT(15)CAC(3’)]) ; Phi(fl) values of between 0.06-0.35 were determined for the double-stranded conjugates. The Phi(fl) value (0.17) of thiazole orange, 1-(N,N’-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)- 2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1 : dA(15)(.)d(5’)[CACT(15)CAC(3’)] (1:1)) is much less than that for some of the hybrids of the conjugates.
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 tripler as compared to that formed with the unmodified oligonucleotide.
Perylene has been covalently linked, via polymethylene tethers. to the 5’- and the 3’-ends of an oligopyrimidine sequence. The presence of the polycyclic ligand stabilizes the duplexes and the triplexes formed by the modified oligonucleotides and their single- and double-stranded DNA targets Lis compared to those formed with the parent unmodified oligonucleotide used as reference. Stabilization of the triplex is at its highest when perylene is linked to the 5’-end of the oligonucleotide via a nine-atom size linker. Stabilization of the duplexes is nearly equivalent whatever the position of the substitution (5’ or 3’) and the linker size used to tether both entities. (C) 2001 Elsevier Science Ltd. All rights reserved.
Thiazole orange label was coupled to the eighth phosphate of a pentadeca-2 ’ -deoxyriboadenylate via a phosphoramidate linkage using different linkers. The stereoisomers were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also linked to the tenth phosphate of icosathymidylates in both the a and the ß series via a phosphoramidate linkage. Once again, the thiazole orange-icosathymidylate conjugates were obtained as pure stereoisomers. The binding properties of these oligo-2 ’ -deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. The covalent attachment of the thiazole orange derivatives to the oligoadenylates stabilizes the complexes formed with both the DNA and RNA targets. On the contrary, when the thiazole orange is tethered to the oligo-a -thymidylate or oligo-ß -thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.
"Light-up" probes, icosa-a -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-a -thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes inta 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.
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.
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.
sAmsacrine-4-carboxamide-oligonucleotide conjugates were synthesized and studied for their capacity to form DNA triple helices and to after 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.
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 isostabilized temperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.
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.
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 thy-nine 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. Ln 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 cross-links were examined in yeast cells.
Solid-phase synthesis of 5’-5’-linked oligonucleotides with opposite polarities tethered via nucleic bases has been performed using a modified dinucleoside bearing an H-phosphonate group at the 3’-position of one nucleoside and a dimethoxytrityl group-at the 3’-position of the second nucleoside. This system is aimed at forming a base tetrad at the junction in order to provide better stabilization. The linker used between the two 5’-terminal bases in the same plane involves a triple bond in order to rigidify the junction. The two oligonucleotide :chains are either made of natural nucleosides or one of them is built With N3’ —>P5’ phosphoramidates. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.
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 G(4Et)C 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 chemical precesses involved in the preparation of oligonucleotide-intercalator conjugates are reviewed. Two strategies are used to covalently attach an intercalating agent to oligonucleotides ; it can be either directly incorporated during the oligonucleotide chain assembly or linked to the unblocked oligonucleotide using a specific coupling reaction. The covalent attachment of different intercalating agents to various positions of an oligonucleotide chain : 5’-, 3’-, both 5’- and 3’-ends, internucleotidic phosphate, 1’- or 2’-position of a sugar residue or nucleic base is reported, Studies of the binding of these modified oligonucleotides to their targets are also presented together with some of the unique properties which are conferred upon the oligonuleotides by attachment of an intercalating agent.
Modified G*C, *GC or *G*C base pairs have been incorporated at the 3(rd) and 8(th) positions of a self-complementary decadeoxyoligonucleotide. The influence of these modifications on duplex stabilities has been studied by absorption spectroscopy. It has been found that a few of them have thermal stabilities similar to that of the AT base pair. (C) 1997 Elsevier Science Ltd.
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 (d(4Et)C), which hybridizes specifically with natural dG to give a G(4Et)C base pair whose stability is very close to that of natural AT base pairs, Duplexes built with AT and/or G(4Et)C 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.
Modified G*C, or A*U base pairs have been incorporated at the 4(th) position of a duplex composed with nine base pairs. The influence of these modifications on duplex stabilities has been studied by absorption spectroscopy. It has been found that G(4Me)C has thermal stability similar to that of the A(5Prop)U base pair. Thus duplexes involving G(4Me)C or A(5Prop)U base pairs exhibit the same Tm. (C) 1997 Elsevier Science Ltd.
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.
Two new modified deoxyguanosine derivatives linked through their C-6 position to a psoralen and an acridine derivative have been synthesized and incorporated into oligonucleotide chains. Difficulties observed dining the purification of oligonucleotides containing a stretch of G and one method used to solve this problem are discussed.
Two peptide-oligonucleotide conjugates 10a and 10b, containing as their peptide moiety the active site mimic of ribonuclease A (HGH motif) and the Cu (ID complexing metallopeptide (GGH motif) respectively, have been synthesized by original on-line solid phase synthesis using pentafluorophenyl active esters and Boc-His(Tos)-OH. Mild basic conditions for the final deprotection and reversed-phase purification afforded the pure hybrid molecules in good yields. The conjugates have been characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
2-Methoxy-6-chloro-9-aminoacridine has been coupled via a polymethylene linker to various positions of an oligonucleotide chain : the 3’-position, using a new universal support, the 5’-position, and both 5’- and 3’-positions via a phosphate. The intercalating agent was also linked to the oligonucleotide chain via an internucleotide phosphorothiolate. The mixture of diastereoisomers was obtained as well as each pure R(p) and S-p isomer. Finally, the acridine moiety was introduced to the 5-position of the deoxyuridine. The binding properties of these oligonucleotide-acridine conjugates with their DNA counterparts have been studied by absorption spectroscopy.
Peptide-oligonucleotide hybrid molecules 6 and 7 have been synthesized by on-line solid phase synthesis using allyloxycarbonyl (Alloc) side chain protection for serine. The conjugates have been characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The interaction of conjugates 6 and 7 with a complementary sequence has been studied by absorption spectroscopy.
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 C-3-U-3 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.
The solid-phase preparation of oligodeoxyribonucleotides covalently linked via nucleic bases with normal (3’-5’) or inverted (5’-5’) polarities is reported. The key-step of these syntheses is the preparation of the tethered dimers.
Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G double right arrow 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 tire reactivity of the activatable dodecamers (cleavage oil 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).
A procedure for the preparation of oligonucleotide-peptide hybrid molecules by means of automated synthesis is described. The conjugates have been assembled on silica supports including CPG (Controlled Pore Glass) and Fractosil supports. The novel N-epsilon-lysine protecting group, 1-(3,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) was used.
The solid-phase synthesis of 5’-5’-linked oligonucleotides tethered via nucleic bases and with opposite polarities has been performed using a modified dinucleoside bearing a phosphoramidite group at the 3’-position of one nucleoside and a dimethoxytrityl group at the 3’-position of the second nucleoside.
The interaction between oligo-a-thymidylates covalently linked to an intercalating agent (an acridine derivative) and their complementary sequences containing ß-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.
Partially phosphate-methylated oligodeoxyribonucleotides have been synthesized on an oxalyl-CPG derivatized support using an isopropoxyacetyl group for the protection of the exocyclic amine of the nucleic bases. Hybridization properties with the target sequence have been studied by absorption spectroscopy.
Solid-phase preparation of oligo-a-deoxyribonucleotides attached to intercalator, chemically or photochemically reactive groups through either their 5’- or 3’-ends, including use of the 5-methyl-a-deoxycytidine.
The solid-phase preparation of oligonucleotides tethered via nucleic bases and with opposite polarities has been performed starting from a bridged dimer bound to a support.
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.
The solid-phase preparation of oligodeoxyribonucleotides attached to intercalator or reactive groups through their 5’- and (or) 3’-ends is reported. These syntheses implicate the introduction of suitable masked functional groups at the 5’-end of the oligonucleotide by the intermediate of their phosphoramidite derivatives or at the 3’-end of the oligonucleotide using modified solid supports. After full deblocking, the functional groups (phosphate, thiophosphate, primary amine or thiol) can be reacted with the suitable reactive group involved in the chosen ligand. These methods allow the preparation of heterobifunctional derivatized oligodeoxyribonucleotides.
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.
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.
The use of modified supports involving 2,2’-dithiodiethanol allows solid-phase synthesis of oligonucleotides derivatized at their 3’ end with an acridine or a phenanthroline derivative or with phosphate, thiophosphate and amino group.
Modified deoxynucleosides 2’-deoxy-ß-L-uridine, ß-L-thymidine, a-L-thymidine, 2’-deoxy-ß-L-adenosine and 2’-deoxy-a-L-adenosine were synthesized and assembled as homooligomers, respectively : octa-ß-L-deoxyuridylates, octa ß-L and a-L-thymidylates and tetra ß-L and a-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 ß-L-d(Up)8m5Acr, ß-L-(Tp)8m5Acr, a-L-(Tp)8m5Acr did not interact with poly(rA) and poly(dA), ß-L-d(Ap)4m5Acr and a-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-ß-D-thymidylate and tetra ß-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. ß-L- and a-L-oligomers demonstrate a high resistance toward nuclease digestion.
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.
Starting from each pure stereoisomer of neopentylphosphothionotriester dimer, octathymidylates involving alternating stereoregular neopentylphosphothionotriester-phosphodiester linkages and covalently linked to an acridine derivative at their 3’-end have been synthesized. Their stability towards nuclease degradation was studied by comparison with an octanucleotide-acridine conjugate containing only phosphodiester groups.