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

181

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...

Section: Resultsmentioning

confidence: 76%

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

Asseline¹,

Delarue²,

Lancelot³

et al. 1984

181

“…Oligo(dT)"s were labeled with 6-(phenylthio)-9-(w-hydroxyalkylamino)-2-methoxyacridine by a modification (9) of the method of Asseline et al (10). These derivatized oligo(dN)s emitted fluorescence at >520 nm when excited at 488 nm by an argon ion laser.…”

Section: Methodsmentioning

confidence: 99%

Characterization of oligonucleotide transport into living cells.

Loke

Stein²,

Zhang³

et al. 1989

702

262

Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptormediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.When oligodeoxynucleotides [oligo(dN)s] complementary to the 5' region of c-myc mRNA are added to cells in culture, c-myc protein synthesis is specifically inhibited (1)(2)(3)(4)(5). Furthermore, addition ofantisense oligo(dN)s to cultures inhibits intracellular viral replication (6)(7)(8) Fig. 1 Top depicts a typical fluorescence histogram comparing cells incubated with no oligo(dN) to those incubated for 24 hr with either acridine-labeled oligo(dN) alone or in the presence of excess unlabeled oligo(dN). Intracellular localization of fluorescence was confirmed by fluorescence microscopy of similarly treated cells (Fig. 1 Middle and Bottom). When we examined the rates of accumulation of variously sized acridine-labeled oligo(dN)s we found that the accumulated intracellular fluorescence after incubation of HL60 cells with 12.5 AtM acridine-labeled oligomers [ranging in size from oligo(dT)3 to oligo(dT)20] increased gradually, plateauing within -50 hr after addition of acridine-labeled oligo(dN) to the culture medium ( Fig. 2A). This is in contrast to the 90 min required

“…3). This increased stability was due to the intercalation ofthe acridine derivative in the hybrid duplex (6,8). The difference between the elution temperatures for the decamers r2 and r2-Acr (11.5°C) was higher than for the pentadecamers r3 and r3-Acr (40C).…”

mentioning

confidence: 93%

Specific inhibition of mRNA translation by complementary oligonucleotides covalently linked to intercalating agents.

Toulmé¹,

Krisch²,

Loreau³

et al. 1986

102

Synthetic oligodeoxynucleotides that are covalently linked at their 3' end to an acridine derivative and are complementary to the repeated sequence UUAAAU-UAAAUUAAA adjacent to the ribosome binding site of the gene 32-encoded mRNA from phage T4 have been used to regulate the synthesis of gene 32-encoded protein in vitro. These modified, synthetic oligonucleotides specifically block the translation of gene 32-encoded mRNA with a higher efficiency than the homologous unsubstituted oligonucleotides. The inhibition produced by these short "anti-messengers" is due to the formation of specific mRNA-oligodeoxynucleotide hybrids that are stabilized by the intercalation of the acridine ring in the RNADNA duplex.