Capillary electrophoresis of RNA oligonucleotides: Catalytic activity of ahammerhead ribozyme

Ribozymes are sequences of catalytic RNA that are being evaluated as possib le antisense therapeutics. This paper describes how capillary electrophores is (CE) could be used to measure the catalytic rate of a synthetic hammerhe ad ribozyme in cleaving its substrate, This substrate was a synthetic full- RNA 17-mer, whereas the ribozyme was made up of a mixture of 37 2'-OH and 2 '-OCH3 RNA nucleotides. After experimental conditions to exclude ribonuclea se contamination were successfully met, different CE modes were tried out t o separate the ribozyme from its substrate. Only the combination of chemica l and thermal denaturation was adequate to disrupt strong secondary structu res and to inhibit comigration of the two molecules. Cleavage kinetics were measured by continuous injection from the reaction vial into a polymer-fil led capillary, and by determination of the area of the shrinking substrate peak. Compared to the well-established slab gel electrophoresis, CE is at l east one order of magnitude faster, may be completely automated, allows eas ier and more precise quantitation of results, and, due to the small scale a nd self-contained nature of the apparatus, reduces health risks from danger ous chemicals, Unfortunately, UV detection in a 100-mu m internal diameter capillary lacked the sensitivity to perform assays in the nanomolar range, which was necessary for a full Michaelis-Menten analysis. (C) 1999 Academic Press.