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 triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex 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 triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex 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 triplex 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 triplex 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, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.
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Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM Unité 201-CNRS UA 481, 43 rue Cuvier, 75231 Paris Cedex 05, France.
François
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JC
Hélène
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D000166:Acridines; D000225:Adenine; D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D006147:Guanine; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011687:Purines; D013056:Spectrophotometry, Ultraviolet; D013873:Thionucleotides; D013941:Thymine
D000166:Acridines; D009838:Oligodeoxyribonucleotides; D011687:Purines; D013873:Thionucleotides; D006147:Guanine; D004247:DNA; D000225:Adenine; D013941:Thymine