Co-operative non-enzymatic base recognition III. Kinetics of the helix—coil transition of the oligoribouridylic · oligoriboadenylic acid system and of oligoriboadenylic acid alone at acidic pH

Abstract

The kinetics of the transition from a double helix to a random coil have been investigated for oligoadenylic · oligouridylic acid at neutral pH and for oligoadenylic acid at acidic pH. The oligoadenylic—oligouridylic acid system has been studied by the temperature-jump and the stopped-flow method. Two different types of helix—coil transitions have been observed: (1) the double helix—coil transition, characterized by a single relaxation time (except in the case of long chains), (2) the triple helix—coil transition with a relaxation process which is slower by a factor of about 100. These processes were followed by characteristic changes in the u.v. spectrum at different A to U ratios. Conditions were found where the double helix—coil transition could be observed without interference from triple-stranded helices. The kinetics of the helix—coil transition have been measured for the chain lengths 8, 9, 10, 11, 14 and 18. The rate constants of helix formation are about 10 6 M −1 sec −1 and are associated with negative apparent activation enthalpies of about −9 kcal./mole. The rates of dissociation at a given temperature become smaller with increasing chain length. At constant chain length the rates increase with temperature. The corresponding activation enthalpies are larger for longer chains ( N = 8, AE D = 30 kcal./mole; N = 18, AE D = 93 kcal./mole). At greater chain lengths an additional relaxation process is observed. This process is due to the unzippering equilibrium at the helix ends and is too fast for an analysis by the temperature-jump method. Some stopped-flow measurements with the tetradeca- and the octadeca-nucleotides also demonstrate that the helix formation proceeds as a second-order reaction and that the rate of recombination is associated with a negative activation enthalpy of about −9 kcal./mole. The helix—coil transition of an oligoadenylic acid at acidic pH can be characterized by a single relaxation process. The formation of the helix is second order with a rate constant of 10 6 M −1 sec −1, the value being almost independent of the chain length. The rate of recombination decreases as the temperature increases corresponding to an activation energy of about −6 kcal./mole. The measurements with the oligoadenylic acid system for different chain length have been performed at three degrees of transition. The rates of dissociation at a given degree of transition are the same for all chain lengths. Increasing temperatures lead to increasing rates of dissociation; the activation enthalpies increase with the length of the oligonucleotide. A detailed analysis of these results is given with an elucidation of the elementary steps. It is concluded that the formation of a helical nucleus is the rate-determining step of recombination. From the values of the activation enthalpies it can be shown that three A · U base pairs form a stable nucleus. According to this result, at least two different nucleation parameters are needed: one for the first base pair and another one for the second pair. The elementary step of base pair formation adjacent to a nucleus is shown to proceed with a rate constant of about 10 7 sec −1. The demonstration of a nucleation length consisting of three base pairs is interesting with respect to the evolution of the triplet genetic code. A codon-anticodon interaction via triplets provides a combination of accurate recognition (stable pairing) with high dynamic flexibility.