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 o f the initial structure. To understand the mechanism of this type of kineti cs 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 r ate is a combination of two kinetic pathways: dissociative and sequential d isplacement. The dissociative pathway occurs by the spontaneous dissociatio n of the initial duplex followed by association of the target and unlabeled probe. The sequential displacement pathway requires only the partial melti ng of the initial duplex to allow for the formation of a branched nucleatio n complex with the unlabeled probe, followed by the complete displacement o f the labeled probe by migration of the branch point. The contribution from the dissociative pathway is predominant at temperatures close to the melti ng point of the labeled probe, whereas the contribution from the displaceme nt 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 prob e and a structured region of a target molecule occurs mainly by the sequent ial-displacement mechanism. (C) 2000 Academic Press.