Probing the energetics of oligo(dT).poly(dA) by laser cross-linking.

Abstract

Experimentally determined changes in free energy (delta G(o)) for thymine-thymine interactions occurring in oligo(dT).poly(dA) are dependent on the method used for preparation of the double-stranded template. A rapid laser cross-linking technique was used to examine the equilibrium between oligomers of (dT) bound to either poly(dA) or poly(rA). The single-pulse (4-6 nsec) ultraviolet laser excitation of these polynucleotides causes pyrimidine dimer formation between contiguous oligo(dT) molecules, resulting in a "ligation" of the oligomers. Analysis of the resulting data using standard binding isotherms allowed determination of the degree of cooperativity existing between oligomers. Using the cooperativity, delta G(o), delta H(o), and delta S(o) are calculated, thereby providing thermodynamic parameters for this interaction. The measured cooperativity of oligo(dT) molecule interactions allows direct calculation of the number of 3' ends available as nicked structures or the number of 3' ends associated with gaps for oligo(dT).poly(dA) when used as a substrate for DNA synthesis.