Kinetic study of double-helix formation and double-helix dissociation of polyadenylic acid

Abstract

The changes in the secondary structure of polyadenylic acid [poly(A)], caused by variations of pH at constant temperature, have been investigated. The position of the equilibrium 2 single-strands ⇌ double-strand is shifted towards the left-hand side by an increase in the ionic strength of the solution, and for constant values of the latter, by the addition of small amounts of divalent transition metal ions.

The kinetics of the conformational changes have been investigated by T-Jump, stopped-flow and classical spectrophotometric methods. With the latter two techniques the reactions were initiated by changes in solution pH. The process of double-helix dissociation was found to involve at least three first-order steps. The process of double-helix formation begins with a second-order step leading, via a labile steady-state intermediate, to a multitude of mismatched double-strands. These, in turn, evolve to the final, completely formed double-helical form according to a series of first-order steps. The value of the second-order rate constant (10⁵ dm³ mol^–1 s^–1) is similar to those found for the double-helix formation reactions of oligonucleotides. Increases in pH from 4 to 5.9 were used to study the conversion of a metastable double-stranded form of poly(A) into a form that is to be considered the most stable under the experimental conditions. This rearrangement process occurs through a series of three first-order steps, like the (much faster) process of double-helix dissociation.