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.

Synthesis and binding properties of oligo-2'-deoxyribonucleotides covalently linked to a thiazole orange derivative.

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 alpha and the beta 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-alpha-thymidylate or oligo-beta-thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.

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.

Modification of the 5' terminus of oligonucleotides for attachment of reporter and conjugate groups.

Reporter and conjugate groups can be added directly to the 5' terminus of oligonucleotides by appropriate modification. Conjugate groups can be used to increase the affinity of complementary strands, induce irreversible modification of target sequences, or enable sequences to recognize and permeate target cell membranes. This overview discusses the 5' modifications that can be used and strategies for the covalent attachment of ligands to the modified oligonucleotides. Step-by-step protocols for attachment of conjugate groups are given elsewhere in the series.

Direct attachment of conjugate groups to the 5' terminus of oligodeoxyribonucleotides.

This unit gives protocols for the attachment of intercalating and photoreactive conjugate groups to oligodeoxyribonucleotides. Protocols are given for acridine- and psoralen-conjugated oligonucleotides, and include attachment of the linker, preparation of the phosphoramidite, coupling to the oligonucleotide, deprotection, purification, and characterization.

Incorporation of halogenoalkyl, 2-pyridyldithioalkyl, or isothiocyanate linkers into ligands.

Ligands can be introduced at the 5' terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5' terminus of the oligonucleotide. These are then reacted to give the ligand-oligonucleotide conjugate. The addition of appropriate linkers to ligands is described in this unit. 5'Modification of the oligonucleotide and the final reaction that produces the ligand-conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite- or H-phosphonate-mediated oligonucleotide synthesis.

Modification of the 5' terminus of oligodeoxyribonucleotides for conjugation with ligands.

Ligands can be introduced at the 5' terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5' terminus of the oligonucleotide. These are then reacted to give the ligand-oligonucleotide conjugate. This unit describes the addition of carboxylated and aminoalkylated linkers, and phosphorothioate, phosphate, and masked thiol groups to the 5' terminus of an oligonucleotide. The addition of linkers to ligands and the final reaction that produces the ligand-conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite- or H-phosphonate-mediated oligonucleotide synthesis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative: new study of the influence of the dye attachment site.

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 Rp and Sp 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.

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.

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.

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.