A 15-Base Acridine-Conjugated Oligodeoxynucleotide Forms Triplex DNA with Its IL-2Rα Promoter Target with Greatly Improved Avidity

Abstract

Attachment of 6,9-diamino-2-methoxyacridine to the 5' end of a purine-rich oligodeoxynucleotide targeting a 15 bp oligopurine oligopyrimidine stretch in the promoter region of the interleukin-2 receptor alpha chain (IL-2R alpha) gene results in an approximately 500-fold increase in its triplex forming avidity as determined by both band shift assay and DMS footprinting (Kd lowered from 2.5 microM to 5 nM). This oligonucleotide participates in Mg(2+)-dependent three-stranded DNA formation in which it is oriented antiparallel relative to the purine strand of the target duplex as determined by acridine moiety sensitized photoreactivity with the target duplex DNA. The oligonucleotides used in these studies were synthesized with a 3-amino-2-hydroxypropyl group at the 3' end to protect against exonucleolytic degradation for future in vivo applications. The 3'-amino group underwent partial removal, probably during the NaOH deprotection step. Both the 3'-amino and the 3'-free forms of the oligo have the same binding avidity and specificity. The interaction of the third strand with its target is sequence specific and can be essentially abolished by a point G-->T transversion 4 bases away from the 3' end of the target oligopurine block or severely reduced by other mutations within the target duplex. Thus, the attachment of the acridine moiety to the 5' end of the oligonucleotide does not seem to substantially compromise the sequence specificity of binding. Additionally, the oligonucleotide composed of G and A nucleotides was found to be superior to the oligonucleotide containing G and T residues since the difference in avidity of binding to the same target site was 17-fold.

Targeting DNA with Triplexes

“…Attachment to the 5'-end has a greater effect than at the 3'-end [130,134], possibly reflecting a structural difference between the 5'-and 3'-triplex-duplex junctions, since the junction at the 5'-end of the third strand is thought to present a strong intercalation site [135,136]. Most of these studies have used parallel, pyrimidine-containing oligonucleotides, though they have also been used to stabilise antiparallel GA and GT-containing triplexes [92,[137][138][139][140] as well as alternate strand triplexes containing both parallel and antiparallel motifs [141]. Attachment generally increases triplex stability by at least 100-fold, though the interaction with related non-cognate sequences is also increased [133].…”

“…Attachment generally increases triplex stability by at least 100-fold, though the interaction with related non-cognate sequences is also increased [133]. However attachment of the acridine does not seem substantially to compromise the sequence specificity of binding [139]. At secondary sites where more than one binding Triplex-binding ligands have also been used for increasing the strength of binding to sites which contain pyrimidine interruptions.…”

“…mode is possible the oligonucleotide appears to bind so as to position the mismatches furthest from the intercalating moiety [133]. For GA-containing oligonucleotides covalent attachment of acridine partially overcomes the inhibitory effect of potassium on triplex formation [138], though GA containing oligonucleotides are still superior to those containing GT-residues [139].…”

Base triplet nonisomorphism strongly influences DNA triplex conformation: Effect of nonisomorphic G∗︁ GC and A∗︁ AT triplets and bending of DNA triplexes

“…Clearly, then, knowledge of the kind of distortions induced by base triplet nonisomorphism would be important in enabling better design of modified bases such as to nullify the D effects (Dt8 and Dr A ) that will optimize/ minimize the destabilizing (mechanistic) effects of base triplet nonisomorphism so as to pave the way for improved stability of the triplex formed. Also, knowledge of the structural details of the grooves of the GA triplex portrayed here may facilitate the design of better ligands 63,64 specific for either the CH or the WH groove of the triplex to enhance the stability of the GA triplex. Thus, structural information of DNA triplexes comprising nonisomorphic base triplets such as reported here would be beneficial in the efficient design of TFOs to regulate gene expression as well as to induce sequence-specific distortions in a target DNA duplex.…”

Enhancement of TFO Triplex Formation by Conjugation with Pyrene <i>via</i> Click Chemistry

“…19) Molecules with non-selective stabilizing effects would represent a powerful tool to promote triplex formation. 20,21) Among these molecules, twisted intercalating nucleic acids (TINA) have been shown to enhance the stability of triplex formation. 22) Our group previously showed that TFOs containing artificial nucleoside analogues, WNA-Et-T, exerted stabilizing effects on all four base pairs (GC, AT, CG and TA base pairs).…”