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.

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.

Design of bifunctional oligonucleotide intercalator conjugates as inhibitors of gene expression.

Oligonucleotide-intercalator conjugates have been designed to control gene expression at the translational and transcriptional level. The intercalator provides an additional binding energy when the oligonucleotide binds either to a complementary sequence on a single-stranded nucleic acid or to a homopurine.homopyrimidine sequence on duplex DNA. The oligonucleotide-intercalator conjugate can arrest translation of a mRNA (the "antisense" strategy); it can block transcription of DNA (the "antigene" strategy). Some of the intercalators that we have chosen can induce irreversible reactions in their target sequence. Here we summarize the reactions that can be targeted to specific sequences of duplex DNA. Phenanthroline induces cleavage of the two strands of duplex DNA in the presence of Cu(II) and a reducing agent. Ellipticine derivatives can be used to photo-induce cleavage. Psoralen derivatives can cross-link the two strands of DNA under near UV irradiation. In all cases the chemical or photochemical reactions are targeted to a specific sequence of duplex DNA.

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: I. A 1H and 31P n.m.r. study.

The structure of an intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex has been investigated by using ultraviolet absorption, circular dichroism, 1H and 31P n.m.r. The binding of cis-DDP does not inhibit the formation of a duplex but it induces a lowering of congruent to 26 degrees C of its melting temperature. A broadening of the 1H spectrum prevents an accurate analysis of the platination site. Nevertheless, by considering its thermal behavior and the number of imino protons a model of structure of the platinated duplex is proposed in which the central C.G. pair is disrupted and a neighboring C.G pair is very accessible or distorted. The environment of two phosphate groups is disturbed by the cis-DDP binding.

Sequence-targeted cleavage of single- and double-stranded DNA by oligothymidylates covalently linked to 1,10-phenanthroline.

The nuclease activity of 1,10-phenanthroline copper ion was targeted to a specific sequence by attachment of the ligand to the 5' or 3' end of octathymidylates. An acridine derivative was also attached to the other end of the oligothymidylate-phenanthroline conjugate. The duplex formed by the oligothymidylate with its complementary sequence was stabilized by intercalation of the acridine derivative. The reaction induced by 3-mercaptopropionic acid led to a very localized cleavage of a 27-nucleotide-long DNA fragment containing a (dA)8 sequence. At high NaCl concentration or in the presence of spermine, cleavage of the single-stranded 27-mer fragment occurred on both sides of the target sequence. This was ascribed to the formation of a triple helix involving two 1,10-phenanthroline-octathymidylate strands that adopt an antiparallel orientation with respect to each other. When a 27-mer duplex was used as a substrate, cleavage sites were observed on both strands. The location of the cleavage sites led us to conclude that the octathymidylate was bound to the (dA)8.(dT)8 sequence in a parallel orientation with respect to the (dA)8-containing strand. This result reflects the ability of thymine to form two hydrogen bonds with an adenine already engaged in a Watson-Crick base pair. This study shows that it is possible to design DNA-binding oligodeoxynucleotides that could selectively recognize and cleave polypurine-polypyrimidine sequences in double-stranded DNA.

Site-specific cleavage of single-stranded and double-stranded DNA sequences by oligodeoxyribonucleotides covalently linked to an intercalating agent and an EDTA-Fe chelate.

An oligodeoxythymidylate, oligo [d(T8)], was covalently linked to an acridine derivative via its 3' end and to EDTA via its 5' end. The octathymidylate was targeted to a single-stranded DNA fragment 27 nucleotides in length containing an octadeoxyadenylate sequence. In the presence of Fe(II) and a reducing agent (dithiothreitol) cleavage reactions were induced in the nucleotide sequence. The extent of the reaction was dependent on oligo concentration, salt concentration and temperature. Dissociation of the complexes at high temperature or low salt concentration abolished the site-specific cleavage reactions. Treatment of the reacted DNA with piperidine or piperidine-formiate strongly enhanced the yield of cleavage reactions demonstrating that damages were induced on nucleic acid bases by the EDTA-Fe complex covalently linked to the octathymidylate. At high salt concentration (1 M NaCl) or in the presence of spermine and ethylene-glycol a triple helix was formed involving the 27-mer DNA fragment and two oligo[d(T8)]. One of the oligo[d(T8)] was bound parallel and the other antiparallel to the oligo[d(A8)] complementary sequence. Cleavage reactions were induced on both sides of this oligo[d(A8)] target sequence. When a 27-mer duplex was used as a target the oligo[d(T8)] was bound in a parallel orientation with respect to the oligo[d(A8)]-containing strand in the major groove of the double helix. Cleavage reactions were induced on the oligo[d(A8)]-containing strand by the EDTA-Fe chelate attached to the 5' end of the oligo[d(T8)].

Sequence-targeted cleavage of nucleic acids by oligo-alpha-thymidylate-phenanthroline conjugates: parallel and antiparallel double helices are formed with DNA and RNA, respectively.

Oligodeoxynucleotides can be synthesized by using the alpha anomers of nucleoside units. Oligo-alpha-deoxynucleotides are resistant to nucleases and could be used to regulate gene expression in vivo. Theoretical calculations were carried out to determine the conformational energy of an oligomeric alpha-beta duplex (dA)5.(dT)5 where the adenosine strand contains natural beta-deoxyribonucleotides and the thymidine strand contains synthetic alpha-deoxyribonucleotides. These calculations predict that in the more stable B-like conformation the two strands of the double helix should run parallel to each other whereas in the more stable A-like conformation the two strands should adopt an antiparallel orientation. In order to test these predictions 1,10-phenanthroline was covalently attached to the 5'-end of an alpha-octathymidylate. In the presence of copper ions and a reducing agent (beta-mercaptopropionic acid), the (phenanthroline)2-copper complex generates OH. radicals that cleave phosphodiester bonds in the complementary sequence to which the alpha-octathymidylate is bound. By use of a 27mer oligo-beta-deoxynucleotide containing an octadeoxyadenylate sequence as a target for the phenanthroline-substituted alpha-(dT)8, cleavage was observed on the 5'-side of the (dA)8 sequence, demonstrating that the alpha-beta DNA-DNA hybrid formed a double helix with parallel orientation of the two strands. The same result was obtained when alpha-(dT)8 was bound to beta-(dA)n with n = 8 or 10. When a beta-oligoriboadenylate was used as a target, cleavage occurred exclusively on the 3'-side of the (rA)8 or (rA)10 sequence, indicating that the alpha-beta DNA-RNA hybrid formed a double helix with an antiparallel orientation of the two strands. When a phenanthroline-substituted beta-octathymidylate was used instead of the alpha-octathymidylate, an antiparallel double helix was formed independently of whether the target beta sequence was a DNA or an RNA.

Targeted cleavage of polynucleotides by complementary oligonucleotides covalently linked to iron-porphyrins.

Oligothymidylates covalently linked to iron-porphyrins were synthesized to target the nuclease activity of Fe-porphyrin to complementary polynucleotides. In the presence of oxygen and a reducing agent, oligo(dT)7 bearing the reactive group attached to the 3'-phosphate was shown to be active in the cleavage of poly(dA) and poly(rA) but not poly(dT). When poly(dA) was used as a matrix, the reaction yield was higher at low temperature where the complexes are stable; upon increasing temperature, the reaction yield decreased in agreement with the dissociation of the oligonucleotide-polynucleotide complex as measured by absorption spectroscopy. Thus, oligonucleotides covalently linked to iron-porphyrin derivatives can be used to cleave selectively the target sequence of the oligonucleotide.