Triplex formation with � anomers of purine-rich and pyrimidine-rich oligodeoxynucleotides. Nucleic Acids Res

Nuclease-resistant a anomers of pyrimidine-rich CT-and purine-rich GA- and GT-containing oligonucleo-tides were investigated for their triplex-forming poten-tial and compared with their corresponding nuclease-sensitive (3 anomers. Both 23mer CT-a and 23mer CT-p had quite similar triplex binding affinities. Synthetic 23mer GT-a oligonucleotides were capable of triplex formation with binding affinities slightly lower than corresponding 23mer GT-p oligonucleo-tides. The orientation of third strand GT-a binding was parallel to the purine strand of the duplex DNA target, whereas the orientation of third strand GT-p binding was found to be antiparallel. Triplex formation with both GT oligonucleotides showed the typical depend-ence on magnesium and temperature. In contrast, 23mer GA-a oligonucleotides did not support triplex formation in either orientation under a variety of experimental conditions, whereas the corresponding 23mer GA-p oligonucleotides demonstrated strong triplex formation in the antiparallel orientation. GA-a oligonucleotides covalently conjugated to acridine were similarly unable to demonstrate triplex formation. GA-a oligonucleotides, in contrast to GT-a oligo-nucleotides, were capable of self-association, detect-able by gel retardation and UV spectroscopy, but competing self-association could not fully account for the lack of triplex formation. Thus for in vivo triplex gene regulation strategies using GT oligonucleotides the non-natural a anomer may be a feasible alternative to the natural p anomer, allowing for a comparable degree of triplex formation without rapid cellular degradation. However, a anomeric inversion does not appear to be a feasible alternative in applications involving GA oligonucleotides