Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative

Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their tripler-forming potential on a 23 bp DNA duplex target, In our system, QA-containing oligophosphorothioates (23mer GA-PS) were capable of tripler formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target, In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support tripler formation in either orientation, whereas the 23mer CT-PO oligophosphodiester demonstrated tripler formation in the antiparallel orientation, GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in tripler stability when phosphorothioate linkages were used, The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated tripler formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide, A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester, This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, tripler-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.