APPLICATION OF A 5'-BRIDGING PHOSPHOROTHIOATE TO PROBE DIVALENT METALAND HAMMERHEAD RIBOZYME MEDIATED RNA CLEAVAGE

This paper describes the preparation and application of a chimeric DNA /RNA oligonucleotide that contains a single 5'-bridging phosphorothioa te linkage adjacent to a ribonucleotide and embedded in an otherwise a ll-DNA sequence. The influence of pH, divalent metal cation, hybridiza tion, and secondary structure on the susceptibility of the thio linkag e towards transesterification is investigated in an effort to better u nderstand the metal-phosphorothioate interactions and the basis for ca talysis. In addition to the chemical cleavage, we have examined the ha mmerhead ribozyme mediated cleavage of the 5'-bridging phosphorothioat e linkage specifically to test the hypothesis that the ribozyme employ s a second metal cofactor, which functions as a Lewis acid, to catalyz e transesterification. The results of our kinetics experiments do not support this double-metal model. (C) 1997 Elsevier Science Ltd.