The kinetics of oligonucleotide replacements

The formation of a duplex between two nucleic acid strands is restricted if one of the strands forms an intra- or intermolecular secondary structure. The formation of the new duplex requires the dissociation and replacement of the initial structure. To understand the mechanism of this type of kinetics we studied the replacement of a labeled DNA oligonucleotide probe bound to a complementary DNA target with an unlabeled probe of the same sequence. The replacement kinetics were measured using a gel-shift assay for 12, 14 and 16-nucleotide probes as a function of temperature and concentration of the unlabeled probe. The results demonstrate that the overall replacement rate is a combination of two kinetic pathways: dissociative and sequential displacement. The dissociative pathway occurs by the spontaneous dissociation of the initial duplex followed by association of the target and unlabeled probe. The sequential displacement pathway requires only the partial melting of the initial duplex to allow for the formation of a branched nucleation complex with the unlabeled probe, followed by the complete displacement of the labeled probe by migration of the branch point. The contribution from the dissociative pathway is predominant at temperatures close to the melting point of the labeled probe, whereas the contribution from the displacement pathway prevails at lower temperatures and when the concentration of the replacing unlabeled probe is high. The results show that at physiological conditions, duplex formation between a single-stranded oligonucleotide probe and a structured region of a target molecule occurs mainly by the sequential-displacement mechanism.