Kinetic property of bulged helix formation: Analysis of kinetic behavior using nearest-neighbor parameters

Bulge structure is one of the well-known and important nucleic acid seconda ry structures containing unpaired nucleotides. Although the thermodynamic p roperty has been thoroughly investigated, the detailed kinetic property of the bulged helix formation is still unknown. We now investigated the helix formation mechanism for bulged helices using temperature-jump experiments. The activation energy for the duplex association (Ea+1) obtained from the t emperature dependence of the rate constants showed that the Ea+1 value depe nded on both the bulged nucleotide and its flanking base pairs. The activat ion energy for duplex dissociation (Ea-1), however, did not always depend o n the bulged nucleotide. In the case of d(TAGCGTTATAA)/d(ATCCAATATT) with o ne C bulge (GCG-bulge helix) and d(TAGAGTTATAA)/d(ATCCAATATT) with One A bu lge (GAG-bulge helix), that had different bulged nucleotides and the same f lanking base-pairs, the Ea+1 value of -13.5 kcal/mol for the GCG-bulge heli x was 11.9 kcal/mol mure negative than the value of -1.6 kcal/mol for the G AG-bulge helix. The Ea-1 value of 50.9 kcal/mol for the GCG-bulge helix was , however, close to 48.7 kcal/mol of the GAG-bulge helix. These data indica te that the rate-limiting steps for both the GCG-bulge and GAG-bulge helice s are likely to be the same step. Furthermore, since it was known that full y matched helix formations can be estimated using nearest-neighbor paramete rs [Williams, A. P.; Longfellow, C. E.; Freier, S. M.; Kierzek, R.; Turner, D. H Biochemistry 1989, 28, 4283-4291], the kinetic data for a bulged heli x were also analyzed and found analogous to the fully matched double helice s. As a result, we found that the energy diagrams of the helix formations f or bulged helices could be estimated using nearest-neighbor parameters for the DNA/DNA helix. According to these diagrams, the rate-limiting step for bulged helices can be considered to be the formation of four or five base p airs containing one bulged nucleotide. These results showed that the analys is of the kinetic behavior using nearest-neighbor parameters is a feasible approach and makes possible the understanding and prediction of folding in the unpaired regions of nucleic acids.