QUANTITATION OF IN-VITRO ACTIVITY OF SYNTHETIC TRANS-ACTING RIBOZYMESUSING HPLC

The cleavage activity of synthetic ribozymes needs to be characterized by reliable and rapid methods. A chromatographic method to simultaneo usly quantitate the amounts of substrate, cleavage fragments and riboz yme is described. The method allows the rapid normalization of analyti cal data because the sum of the 260-nm peak areas of remaining substra te and obtained fragments is essentially equal to the initial substrat e peak area. Moreover, the simultaneous determination of the ribozyme content improves the accuracy of the evaluation of kinetic parameters compared with conventional densitometric methods. Finally, the charact erization of two different hammerhead motifs indicated that the method is suitable for a rapid screening of synthetic ribozyme activity.