Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

The binding of a series of PNA and DNA probes to a group of unusually stabl e DNA hairpins of the tetraloop motif has been observed using absorbance hy pochromicity (ABS), circular dichroism (CD), and a colorimetric assay for P NA/DNA duplex detection. These results indicate that both stable PNA-DNA an d DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degreesC lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/ hairpin interactions are considered in the scope of these studies. Secondar y structures in both target and probe molecules are shown to depress the me lting temperatures and free energies of the, probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold sl ower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe se condary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.