Quantitation of In Vitro Activity of Synthetic Trans-Acting Ribozymes Using HPLC | Zendy

Lorenzo Citti | Zendy; Laura Boldrini | Zendy; Silvio Nevischi | Zendy; L. Mariani | Zendy; Giuseppe Rainaldi | Zendy

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Quantitation of In Vitro Activity of Synthetic Trans-Acting Ribozymes Using HPLC

Author(s) -

Lorenzo Citti,

Laura Boldrini,

Silvio Nevischi,

L. Mariani,

Giuseppe Rainaldi

Publication year - 1997

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/97235st07

Subject(s) - ribozyme , cleavage (geology) , chemistry , chromatography , normalization (sociology) , substrate (aquarium) , in vitro , hairpin ribozyme , high performance liquid chromatography , biochemistry , biology , rna , gene , paleontology , ecology , sociology , fracture (geology) , anthropology

The cleavage activity of synthetic ribozymes needs to be characterized by reliable and rapid methods. A chromatographic method to simultaneously quantitate the amounts of substrate, cleavage fragments and ribozyme is described. The method allows the rapid normalization of analytical data because the sum of the 260-nm peak areas of remaining substrate and obtained fragments is essentially equal to the initial substrate 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 characterization of two different hammerhead motifs indicated that the method is suitable for a rapid screening of synthetic ribozyme activity.

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