Application of a 5'-bridging phosphorothioate to probe divalent metal and hammerhead ribozyme mediated RNA cleavage

This paper describes the preparation and application of a chimeric DNA/RNA oligonucleotide that contains a single 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide and embedded in an otherwise all-DNA sequence. The influence of pH, divalent metal cation, hybridization, and secondary structure on the susceptibility of the thio linkage towards transesterification is investigated in an effort to better understand the metal-phosphorothioate interactions and the basis for catalysis. In addition to the chemical cleavage, we have examined the hammerhead ribozyme mediated cleavage of the 5'-bridging phosphorothioate linkage specifically to test the hypothesis that the ribozyme employs a second metal cofactor, which functions as a Lewis acid, to catalyze transesterification. The results of our kinetics experiments do not support this double-metal model.