Selective circular oligonucleotide probes improve detection of point mutations in DNA

The synthesis of disulfide-cross-linked circular oligonucleotides, employing two different approaches, was accomplished. Several circular oligomers, which bear a C(5)-aminoalkyl-tethered thymidine unit, were labeled with photoluminescent europium(III) chelates. All circular structures were thoroughly characterized with denaturing PAGE and electrospray-ionization mass spectrometry. It was demonstrated that the disulfide cross-linking, resulting in circularization, considerably increases the enzymatic stability of phosphodiester oligonucleotides. In addition, UV melting experiments, followed, where possible, by extraction of thermodynamic parameters, revealed that several circular oligomers appear to be more selective towards their complementary targets than their corresponding linear precursors. Finally, the mixed-phase hybridization experiments have demonstrated that use of circular probes indeed improves the selectivity in the detection of DNA point mutations.