Synthesis and triplex-forming properties of cyclic oligonucleotides with (G,A)-antiparallel strands.

Cyclic oligonucleotides carrying an oligopurine Watson-Crick sequence linked to the corresponding (G,A)- and (G,T)-antiparallel strands were prepared by nonenzymatic template-assisted cyclization of phosphorylated precursors. Cyclization was attempted using 3'-phosphate and 5'-phosphate linear precursors with carbodiimide or BrCN activation. The best results were obtained with the 5'-phosphorylated precursors and carbodiimide activation. Cyclic oligonucleotides bind polypyrimidine target sequence by formation of antiparallel triplexes. We have used UV and circular dichroism (CD) spectroscopy to analyze triplexes formed by cyclic oligonucleotides carrying G and A in the reverse-Hoogsteen strand. The relative stability of the triplexes formed by cyclic and linear oligonucleotides with a common polypyrimidine target was determined by melting experiments. The most-stable triplexes were formed by the cyclic oligonucleotide, followed by the unphosphorylated and phosphorylated oligonucleotide precursors, and, finally, the corresponding hairpin. Although the differences in binding affinity between cyclic oligonucleotides and their corresponding linear precursors are small, the use of cyclic oligonucleotides offers a clear advantage over conventional duplex recognition.