The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides

Abstract

Oligonucleotides are being developed to selectively inhibit gene expression at the translational level (antisense oligonucleotides) and at the transcriptional level (anti-gene oligonucleotides). This review deals with the anti-gene strategy whereby an oligonucleotide binds to the major groove of double helical DNA where it forms a local triple helix. The molecular mechanisms for DNA recognition by triple helix formation are discussed together with some of the rules presently available to design the sequence and orientation of the triple helix forming oligonucleotide. Triplex stability can be enhanced by covalent attachment of an intercalating agent to the terminal nucleotide of the oligonucleotide. The intercalating agent can be used to induce irreversible reactions in the target sequence: double strand cleavage by a phenanthroline-Cu chelate in the presence of a reducing agent, photo-induced cleavage by ellipticine derivatives, photo-induced cross-linking of the two DNA strands by psoralen... Triple helix-forming oligonucleotides can be used to control gene expression at the transcriptional level. Inhibition of binding of transcription activating factors by triplex formation modulates the level of transcription of the target gene. Binding of a triplex-forming oligonucleotide immediately downstream of the RNA polymerase binding site can inhibit transcription initiation as shown with the E. coli beta-lactamase gene. Studies with cells in culture show that triple helix formation may occur in the intracellular environment and consequently leads to transcription inhibition. This inhibitory effect can be made irreversible by using, e.g., psoralen-substituted oligonucleotides. Oligonucleotides synthesized with the alpha-anomers of nucleotide units are resistant to nucleases and still form triple helices with double-stranded DNA. Oligo-[alpha]-deoxynucleotides can be derived by stabilizing (intercalating) agents or reactive groups (cleaving reagents, cross-linkers ...). The results presently available provide a rational basis for the development of new tools for cellular biology and of new therapeutical approaches to selectively control gene expression at the transcriptional level.