Nucleic acids can be selectively recognized by a large number of natural and synthetic ligands. Oligonucleotides provide the highest specificity of recognition. They can bind to a complementary single-stranded sequence by forming Watson-Crick hydrogen bonds. They can also recognize the major groove of double-helical DNA at specific sequences by forming Hoogsteen or reverse Hoogsteen hydrogen bonds with purine bases of the Watson-Crick base pairs, resulting in a triple helix. Triple-helix formation through oligonucleotide binding to DNA is a sequencespecific interaction involving primarily homopurine . homopyrimidine sequences in the double-helical target. Extending the range of recognition sequences remains a challenge to chemists. Both thermodynamic and kinetic parameters for triplex formation have been determined. These parameters indicate, for example, that triple-helix formation is a much slower process than duplex formation. Nuclease-resistant oligonucleotides synthesized with the aanomers of nucleosides (instead of the natural p-anomers) also form triple helices with doublestranded DNA. Triple-helix-forming oligonucleotides can be modified, for example, by attaching DNA intercalating agents to enhance their binding affinity. They may also be modified with reagents that induce irreversible reactions in their target sequence upon chemical or photochemical activation. Thus, artificial nucleases can be developed with very high sequence specificity on megabase-size DNA. Furthermore, triple-helix-forming oligonucleotides can be used to selectively control gene expression. When bound to the regulatory region(s) of specific genes they may prevent activation (or repression) of transcription. When binding occurs near or downstream from the transcription initiation site, elongation of the transcript may be inhibited. Therefore, the potential exists for developing new gene-blocking agents with therapeutic applications in the treatment of gene disorders.

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [alpha]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[alpha]-thymidylate: a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[alpha]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[alpha]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [alpha]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.

On the basis of the structure of DNA-psoralen bis adducts (formed by psoralen with two thymines on opposite strands), a psoralen-oligonucleotide conjugate was designed to photoinduce a cross-link between the two DNA strands at a specific sequence. Psoralen was attached via its C-5 position to a 5'-thiophosphate group of an 11-mer homopyrimidine oligonucleotide. The 11-mer binds to an 11-base-pair homopurinehomopyrimidine sequence of a DNA fragment, where it forms a triple helix. Upon near-UV-irradiation, the two strands of DNA are crosslinked at the TpA step present at the triplex-duplex junction. The reaction is specific for the homopurine'homopyrimidine DNA sequence and requires both oligonucleotide recognition of the DNA major groove and intercalation of psoralen at the triplex-duplex junction. The yield of the photoinduced cross-linking reaction is quite high (>80%). Such psoralen-oligonucleotide conjugates are probes of sequencespecific triple-helix formation and could be used to selectively control gene expression or to induce site-directed mutations.

Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner. We have recently designed psoralen-oligonucleotide conjugates, which, upon binding to double-stranded DNA sequences via triple helix formation, may be cross-linked in vitro to both strands of the DNA following UV irradiation. A psoralen-oligonucleotide conjugate was targeted to tht promoter of the a subunit of the interleukin 2 receptor (IL-2Ra) gene. The triple helix site overlaps the binding site for the transcription factor NF-KB, which activates transcription from the IL-2Ra promoter. After UV irradiation, the oligonucleotide conXjugate becomes crosslinked to the target site and inhibits transcription of reporter plasmids transfected in live cells. Inhibition is observed when UV-induced cross-linking occurs both in vitro (before transfection) and in vivo (after transfection). We directly demonstrate that this inhibitory effect is due to triple helix formation at the target site, since a mutant of the promoter, to which oligonucleotide binding was inhibited, was not affected by the psoralenoligonucleotide conjugate after UV irradiation. In addition, we demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.

A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural ((3) anomer of thymidine or the synthetic (a) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with a complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA-dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence was observed, generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocrosslinked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both a and (3 oligothymidylates can recognize a DNA double helix containing an oligo(dA)-oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) a oligothymidylates form helices with a complementary singlestranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, (3 oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that it is possible to recognize an oligopurine-oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photochemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.

An acridine derivative was covalently linked to the 5' end of a homopyrimidine oligonucleotide. Specific binding to a homopurine homopyrimidine sequence of duplex DNA was demonstrated by spectroscopic studies (absorption and fluorescence) and by "footprinting" experiments with a copper phenanthroline chelate used as an artificial nuclease. A hypochromism and a red shift of the acridine absorption were observed. Triple-helix formation was also accompanied by a hypochromism in the ultraviolet range. The fluorescence of the acridine ring was quenched by a stacking interaction with a GC base pair adjacent to the homopurine-homopyrimidine target sequence. The intercalating agent strongly stabilized the complex formed by the oligopyrimidine with its target duplex sequence. Cytosine methylation further increased the stability of the complexes. Footprinting studies revealed that the oligopyrimidine binds in a parallel orientation with respect to the homopurine-containing strand of the duplex. The intercalated acridine extended by 2 base pairs the region of the duplex protected by the oligopyrimidine against degradation by the nuclease activity of the copper phenanthroline chelate. Random intercalation of the acridine ring was lost due to the repulsive effect of the negatively charged oligonucleotide tail. Intercalation occurred only at those double-stranded sequences where the homopyrimidine oligonucleotide recognized the major groove of duplex DNA.

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.

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),(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),(CH2)mAcr and poly(rA) involves the formation of n A-T base pairs, where n is the number of thy. mines in the oligonucleotide. The acridme ring intercalates between ANT 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.Molecules with high affinity and base-sequence specificity are required to control gene expression at different levels. Several possibilities may be contemplated depending on whether double-stranded or single-stranded nucleic acids are chosen as targets. For example, the regulation of transcription involves proteins that recognize specific duplex DNA sequences in both prokaryotes and eukaryotes (1, 2). However, there is no general rule yet established that could allow us to build an oligopeptide sequence aimed at recognizing a given base or base pair sequence (3). Moreover, the amino acid side chains that are involved in contacts with base or base pairs are not contiguous in the polypeptide chain.The most obvious candidate to allow for the specific recognition of a nucleic acid fragment is an oligonucleotide with the complementary sequence, provided the nucleic acid bases in the target sequence have their hydrogen bonding sites available. This is obviously so if the target nucleic acid region exists as a single-stranded structure. This might also be true in a duplex structure if a sufficient supplementary energy of interaction were provided either by modifying the oligonucleotide (see below) or by imposing constraints on the duplex structure (e.g., in superhelical DNA or upon binding of melting proteins). Also it must be kept in mind that important regulatory regions in transcription and replication must be transiently opened as observed-e.g., in the complex formed by bacterial RNA polymerase with a promoter (the so-called "open complex") (4) or at the replication fork (5). Actively transcribed genes in eukaryotes also possess regions upstream f...