Very High Affinity DNA Recognition by Bicyclic and Cross-Linked Oligonucleotides

We report the synthesis and DNA incorporation of a novel C-5 thiopropyne-substituted thymidine derivative which can be used to bring about covalent crosslinks between two noncomplementary DNA strands. This modified thymine pairs normally with adenine in duplex DNA and is shown not to be destabilizing to DNA double helices. Placement of the thiol-nucleotide near the center of opposing pyrimidine strands in pyr·pur·pyr triple helices results in crosslinking of the pyrimidine strands under aerobic conditions. Thermal melting studies at neutral pH show that such crosslinked ligands bind complementary purine strands with higher affinity than is possible with simple Watson-Crick recognition alone. In addition, we describe the construction of a triplex-forming circular oligonucleotide which contains a similar disulfide link across the center. This macrobicyclic ligand binds with extremely high affinity and sequence selectivity to a complementary purine DNA strand. The formation of crosslinks across two noncomplementary strands represents a new strategy for increasing affinity and selectivity of DNA recognition.