127] Measurement of nucleic acid hydrogen exchange

Abstract

This chapter discusses the measurement of hydrogen exchange (HX) kinetics of nucleic acids by using the Sephadex and dialysis methods. The movement of H between nucleic acid and H2O solvent is followed with tritium (T) tag. Nucleic acid is initially incubated in THO solvent so that its exchangeable hydrogens come to equilibrium with free, solvent T and H. Unbound T is then removed by Sephadex columns or rapid dialysis to initiate a unidirectional exchange out of bound T. To measure the amount of T remaining bound to the nucleic acid at any time, the T that has exchanged out during the test time interval is removed. In a series of samples taken as a function of time of exchanging out, the ratio of (bound) T level to nucleic acid content is measured, and a plot of these data trace the exchange out kinetics of the nucleic acid. The chapter discusses a rapid dialysis technique that can be used to remove THO from tritiated nucleic acid. The technique, then, is too slow for most experiments with DNA but can be useful for soluble RNA (sRNA) and ribosomes. HX experiments can give information on the amount of structure in nucleic acids in terms of the total number of slowly exchanging hydrogens present. Nucleic acid HX rates are determined by localized opening–closing reactions that, it now seems, are experienced by all macromolecules. These opening–closing reactions may be of basic importance for the functions that nucleic acid molecules perform and for the effect on these functions of various chemical and physical agents.